Modulation of insulin like growth factor I receptor expression

ABSTRACT

The present invention provides compositions and methods for modulating the expression of growth factor gene. In particular, this invention relates to compounds, particularly oligonucleotide compounds, which, in preferred embodiments, hybridize with nucleic acid molecules encoding the Insulin Like Growth Factor I receptor (IGF-I receptor or IGF-IR) and in particular human IGF-IR. Such compounds are exemplified herein to modulate proliferation which is relevant to the treatment of proliferative and inflammatory skin disorders and cancer. It will be understood, however, that the compounds can be used for any other condition in which the IGF-IR is involved including inflammatory condition.

This application is a Continuation application of U.S. patentapplication Ser. No. 10/545,354, entitled MODULATION OF INSULIN LIKEGROWTH FACTOR I RECEPTOR EXPRESSION, filed on Aug. 11, 2005; whichclaims priority to PCT/AU2004/000160, entitled MODULATION OF INSULINLIKE GROWTH FACTOR I RECEPTOR EXPRESSION, filed on Feb. 11, 2004; whichclaims priority to Australian application No. AU 2003902610; entitledMODULATION OF INSULIN LIKE GROWTH FACTOR I RECEPTOR EXPRESSION, filed onMay 27, 2003; which claims priority to Australian application No.AU2003900609; entitled MODULATION OF INSULIN LIKE GROWTH FACTOR IRECEPTOR EXPRESSION, filed on Feb. 11, 2003; all of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

The present invention provides compositions and methods for modulatingthe expression of a growth factor receptor gene. In particular, thisinvention relates to compounds, particularly oligonucleotide compounds,which, in preferred embodiments, hybridize with nucleic acid moleculesencoding the Insulin Like Growth Factor I receptor (IGF-I receptor orIGF-IR). Such compounds are exemplified herein to modulate proliferationwhich is relevant to the treatment of proliferative and inflammatoryskin disorders and cancer. It will be understood, however, that thecompounds can be used for any other condition in which the IGF-IR isinvolved including inflammatory conditions.

BACKGROUND OF THE INVENTION

Bibliographic details of the publications referred to by author in thisspecification are collected at the end of the description.

Reference to any prior art in this specification is not, and should notbe taken as, an acknowledgment or any form of suggestion that this priorart forms part of the common general knowledge in any country.

Psoriasis and other similar conditions are common and often distressingproliferative and/or inflammatory skin disorders affecting or having thepotential to affect a significant proportion of the population. Thecondition arises from over proliferation of basal keratinocytes in theepidermal layer of the skin associated with inflammation in theunderlying dermis. Whilst a range of treatments have been developed,none is completely effective and free of adverse side effects. Althoughthe underlying cause of psoriasis remains elusive, there is someconsensus of opinion that the condition arises at least in part fromover expression of local growth factors and their interaction with theirreceptors supporting keratinocyte proliferation via keratinocytereceptors which appear to be more abundant during psoriasis.

One important group of growth factors are the dermally-derivedinsulin-like growth factors (IGFs) which support keratinocyteproliferation. In particular, IGF-I and IGF-2 are ubiquitouspolypeptides each with potent mitogenic effects on a broad range ofcells. Molecules of the IGF type are also known as “progression factors”promoting “competent” cells through DNA synthesis. The IGFs act througha common receptor known as the Type I receptor or IGF-IR, which istyrosine kinase linked. They are synthesized in mesenchymal tissues,including the dermis, and act on adjacent cells of mesodermal,endodermal or ectodermal origin. The regulation of their synthesisinvolves growth hormone (GH) in the liver, but is poorly defined in mosttissues (Sara, Physiological Reviews 70: 591-614, 1990).

Particular proteins, referred to as IGF binding proteins (IGFBPs),appear to be involved in autocrine/paracrine regulation of tissue IGFavailability (Rechler and Brown, Growth Regulation 2: 55-68, 1992). SixIGFBPs have so far been identified. The exact effects of the IGFBPs isnot clear and observed effects in vitro have been inhibitory orstimulatory depending on the experimental method employed (Clemmons,Growth Regn. 2:80, 1992). There is some evidence, however, that certainIGFBPs are involved in targeting IGF-I to its cell surface receptor.

Skin, comprising epidermis and underlying dermis, has GH receptors ondermal fibroblasts (Oakes et al., J. Clin. Endocrinol. Metab. 73:1368-1373, 1992). Fibroblasts synthesize IGF-1 as well as IGFBPs-3, -4,-5 and -6 (Camacho-Hubner et al., J. Biol. Chem. 267: 11949-11956, 1992)which may be involved in targeting IGF-1 to adjacent cells as well as tothe overlaying epidermis. The major epidermal cell type, thekeratinocyte, does not synthesize IGF-I, but possesses IGF-I receptorsand is responsive to IGF-I (Neely et al., J. Inv. Derm. 96: 104, 1991).

In the last decade, there have been many reports of the use of antisenseoligonucleotides to explore gene function and in the development ofnucleic acid based drugs. Antisense oligonucleotides inhibit mRNAtranslation via a number of alternative ways including destruction ofthe target mRNA through RNaseH recruitment, or interference with RNAprocessing, nuclear export, folding or ribosome scanning. More recently,a better understanding of intracellular sites of action of the variousantisense modalities and improvements in oligonucleotide chemistry haveincreased the number of reports of validated expression inhibition.

In work leading up to the present invention, the inventors focused onthe use of the antisense approach to inhibit the growth of humanepidermal keratinocytes, particularly in human epidermal growthdisorders such as psoriasis. Psoriasis is a common and disfiguring skincondition associated with severe epidermal hyperplasia. Existingpsoriasis therapies are only partially effective, however, treatmentstargeting the epidermis have shown promise (Jensen et al., Br. J.Dermatol. 139: 984-991, 1998; van de Kerkhof, Skin Pharmacol. Appl. SkinPhysiol. 11: 2-10, 1998). One strategy is to develop antisenseinhibitors of IGF-IR expression and to use these to blockIGF-I-stimulated cell division and survival in the epidermis.

The IGF-IR is a tyrosine kinase linked cell surface receptor (Ullrich etal., EMBO J. 5: 2503-2512, 1986) that regulates cell division,transformation and apoptosis in many cell types (LeRoith et al., Endocr.Rev. 16: 143-163, 1995; Rubin and Baserga, Laboratory Investigation 73:311-331, 1995). Human epidermal keratinocytes are highly responsive toIGF-IR activation (Ristow and Messmer, J. Cell Physiol. 137: 277-284,1988; Neely et al., J. Invest. Dermatol. 96: 104-110, 1991; Wraight etal., J. Invest. Dermatol. 103: 627-631, 1994) and several studies pointto an important role for IGF-1R activation in the pathogenesis ofpsoriasis (Krane et al., J. Invest. Dermatol. 96: 419-424, 1991; Kraneet al., J. Exp. Med. 175: 1081-1090, 1992; Ristow, Growth Regul. 3:129-137, 1993; Hodak et al., J. Invest. Dermatol. 106: 564-570, 1996; Xuet al., J. Invest. Dermatol. 106: 109-112, 1996; Ristow, Dermatology195: 213-219, 1997; Wraight et al., J. Invest. Dermatol. 108: 452-456,1997). The IGF-IR has been targeted previously by antisense approachesin fibroblasts, haemopoietic cells and glioblastoma cells to investigateits role in transformation and cell cycle progression (Pietrzkowski etal., Mol. Cell. Biol. 12: 3883-3889, 1992; Porcu et al., Mol. Cell.Biol. 12: 5069-5077, 1992; Reiss et al., Oncogene 7: 2243-2248, 1992;Resnicoff et al., Cancer Res. 54: 2218-2222, 1994).

The identification of propynylated phosphorothioate oligonucleotideshave been reported which are capable of reducing IGF-IR mRNA levels whenefficiently delivered to the keratinocyte nucleus (White et al.,Antisense Nucleic Acid Drug Dev. 10: 195-203, 2000; Wraight et al., Nat.Biotechnol. 18: 521-526, 2000). These oligonucleotides were alsoeffective at reducing IGF-I binding, receptor activation and cellproliferation in vitro and epidermal proliferation in vivo (Wraight etal., 2000, supra).

Propyne-modified phosphorothioate oligonucleotides were selected(Flanagan et al., Nat. Biotechnol. 14: 1139-1145, 1996b; Flanagan andWagner, Mol. Cell. Biochem. 172: 213-225, 1997) because their increasedaffinity for target mRNA allows mRNA inhibition with lowerconcentrations (Wagner et al., 1993, supra) and shorter oligonucleotidelength (Flanagan et al., Nucleic Acids Res. 24: 2936-2941, 1996a) thanunmodified phosphorothioates, theoretically reducing the incidence ofaptameric effects on target cells.

Whilst success has been demonstrated with the propyne-modifiedphosphorothioate oligonucleotides, alternative chemistries need to beconsidered to reduce toxicity, increase stability, increase specificityprofile, improve penetration and/or to enhance potency and biological,chemical or physical properties. Oligonucleotides of alternativechemistries can also provide other advantages including known largescale manufacture, human clinical development knowhow, and/or knownapproval as drugs.

SUMMARY OF THE INVENTION

Throughout this specification, unless the context requires otherwise,the word “comprise”, or variations such as “comprises” or “comprising”,will be understood to imply the inclusion of a stated element or integeror group of elements or integers but not the exclusion of any otherelement or integer or group of elements or integers.

Nucleotide and amino acid sequences are referred to by a sequenceidentifier number (SEQ ID NO:). The SEQ ID NOs: correspond numericallyto the sequence identifiers <400>1 (SEQ ID NO:1), <400>2 (SEQ ID NO:2),etc. A summary of the sequence identifiers is provided in Table 1. Asequence listing is provided after the claims.

The present invention is directed to compounds, especially nucleic acidand nucleic acid-like oligomers, which are targeted to a nucleic acidencoding a growth factor receptor and in particular Insulin Like GrowthFactor I Receptor (IGF-IR), and even more particularly human IGF-IR andwhich modulate the expression of IGF-1R. Pharmaceutical and othercompositions comprising the compounds of the invention are alsoprovided. Further provided are methods of screening for modulators ofIGF-IR gene expression and methods of modulating the expression of theIGF-IR gene in cells, tissues or animals comprising contacting saidcells, tissues or animals with one or more of the compounds orcompositions of the invention. Methods of treating an animal,particularly a human, suspected of having or being prone to a disease orcondition associated with expression of IGF-IR or its ligand, IGF-I, arealso set forth herein. Such methods comprise administering atherapeutically or prophylactically effective amount of one or more ofthe compounds or compositions of the invention to the person in need oftreatment.

The preferred compounds of the present invention are referred to hereinas antisense oligonucleotides or ASOs. The ASOs referred to in thesubject specification are listed in Table 1. The ASOs are identified byan “ISIS” number as well as a SEQ ID number.

One group of particularly preferred ASOs include ISIS 175308 (SEQ IDNO:116), ISIS 175302 (SEQ ID NO:110), ISIS 175314 (SEQ ID NO:122), ISIS175307 (SEQ ID NO:115), ISIS 175317 (SEQ ID NO:125) and ISIS 175323 (SEQID NO:131).

Another group of particularly preferred ASOs include ISIS 323744 (SEQ IDNO:50), ISIS 323747 (SEQ ID NO:53), ISIS 323767 (SEQ ID NO:73), ISIS323762 (SEQ ID NO:68) and ISIS 323737 (SEQ ID NO:43).

An even more particularly preferred ASO is ISIS 175317 (SEQ ID NO:125).

TABLE 1 Summary of nucleic acid molecules TARGET SEQ TARGET % SEQ IDISIS # REGION ID NO SITE SEQUENCE/DESCRIPTION INHIB NO 323695 5′UTRNM_000875.2     25 CCTTTTATTTGGGATGAAAT 50   1 323696 Start CodonNM_000875.2     37 CCAGACTTCATTCCTTTTAT 44   2 323697 Coding NM_000875.2   157 TGATAGTCGTTGCGGATGTC 73   3 323698 Coding NM_000875.2    162GCTGCTGATAGTCGTTGCGG 72   4 323699 Coding NM_000875.2    167CTTCAGCTGCTGATAGTCGT 74   5 323700 Coding NM_000875.2    196CCCTCGATCACCGTGCAGTT 56   6 323701 Coding NM_000875.2    223TTGGAGATGAGCAGGATGTG 65   7 323702 Coding NM_000875.2    228CGGCCTTGGAGATGAGCAGG 66   8 323703 Coding NM_000875.2    233GTCCTCGGCCTTGGAGATGA 71   9 323704 Coding NM_000875.2    238CGGTAGTCCTCGGCCTTGGA 71  10 323705 Coding NM_000875.2    367TTGTAGAAGAGTTTCCAGCC 52  11 323706 Coding NM_000875.2    396TGGTCATCTCGAAGATGACC  5  12 323707 Coding NM_000875.2    401GAGATTGGTCATCTCGAAGA 20  13 323708 Coding NM_000875.2    406TCCTTGAGATTGGTCATCTC 41  14 323709 Coding NM_000875.2    411CAATATCCTTGAGATTGGTC 29  15 323710 Coding NM_000875.2    416AAGCCCAATATCCTTGAGAT 43  16 323711 Coding NM_000875.2    443CCCCCGAGTAATGTTCCTCA 41  17 323712 Coding NM_000875.2    459TCTCAATCCTGATGGCCCCC 56  18 323713 Coding NM_000875.2    527GTTATTGGACACCGCATCCA 31  19 323714 Coding NM_000875.2    532ATGTAGTTATTGGACACCGC 64  20 323715 Coding NM_000875.2    537CCACAATGTAGTTATTGGAC 65  21 323716 Coding NM_000875.2    571CACAGGTCCCCACATTCCTT 42  22 323717 Coding NM_000875.2    576CTGGACACAGGTCCCCACAT 45  23 323718 Coding NM_000875.2    616ATGGTGGTCTTCTCACACAT 69  24 323719 Coding NM_000875.2    621TGTTGATGGTGGTCTTCTCA 66  25 323720 Coding NM_000875.2    626CTCATTGTTGATGGTGGTCT 81  26 323721 Coding NM_000875.2    632GTTGTACTCATTGTTGATGG 73  27 323722 Coding NM_000875.2    637CGGTAGTTGTACTCATTGTT 71  28 323723 Coding NM_000875.2    642AGCAGCGGTAGTTGTACTCA 70  29 323724 Coding NM_000875.2    647GGTCCAGCAGCGGTAGTTGT 60  30 323725 Coding NM_000875.2    652TTTGTGGTCCAGCAGCGGTA 67  31 323726 Coding NM_000875.2    674TGGGCACATTTTCTGGCAGC 57  32 323727 Coding NM_000875.2   1283GGAGTAATTCCCTTCTAGCT 21  33 323728 Coding NM_000875.2   1324TCCCACAGTTGCTGCAAGTT 73  34 323729 Coding NM_000875.2   1678ATGTTCCAGCTGTTGGAGCC 72  35 323730 Coding NM_000875.2   1683CCACCATGTTCCAGCTGTTG 78  36 323731 Coding NM_000875.2   1750GTCCAGGGCTTCAGCCCATG 74  37 323732 Coding NM_000875.2   1786GTGAGGGTCACAGCCTTGAC 59  38 323733 Coding NM_000875.2   1791CCATGGTGAGGGTCACAGCC 78  39 323734 Coding NM_000875.2   1846TTGGTGCGAATGTACAAGAT 61  40 323735 Coding NM_000875.2   2029ATTTTGTCTTTGGAGCAGTA 65  41 323736 Coding NM_000875.2   2203AGGAAATTCTCAAAGACTTT 43  42 323737 Coding NM_000875.2   2290CTGCTTCGGCTGGACATGGT 84  43 323738 Coding NM_000875.2   2295TGTTCCTGCTTCGGCTGGAC 76  44 323739 Coding NM_000875.2   2368CTGCTCTCAAAGAAAGGGTA 58  45 323740 Coding NM_000875.2   2373CCACTCTGCTCTCAAAGAAA  0  46 323741 Coding NM_000875.2   2378GTTATCCACTCTGCTCTCAA 57  47 323742 Coding NM_000875.2   2383TCCTTGTTATCCACTCTGCT 58  48 323743 Coding NM_000875.2   2446TTGCAGCTGTGGATATCGAT 53  49 323744 Coding NM_000875.2   2451CGTGGTTGCAGCTGTGGATA 85  50 323745 Coding NM_000875.2   2456AGCCTCGTGGTTGCAGCTGT 75  51 323746 Coding NM_000875.2   2461TTCTCAGCCTCGTGGTTGCA 62  52 323747 Coding NM_000875.2   2466CCAGCTTCTCAGCCTCGTGG 85  53 323748 Coding NM_000875.2   2471GCAGCCCAGCTTCTCAGCCT 77  54 323749 Coding NM_000875.2   2476GCGCTGCAGCCCAGCTTCTC 71  55 323750 Coding NM_000875.2   2578TTTAAAAAGATGGAGTTTTC  8  56 323751 Coding NM_000875.2   2583GCCACTTTAAAAAGATGGAG 77  57 323752 Coding NM_000875.2   2677TCCTGTCTGGACACACATTC 66  58 323753 Coding NM_000875.2   2791AAGAACACAGGATCTGTCCA 38  59 323754 Coding NM_000875.2   2796CATAGAAGAACACAGGATCT 33  60 323755 Coding NM_000875.2   2992GGAACGTACACATCAGCAGC 36  61 323756 Coding NM_000875.2   3076ACTCCTTCATAGACCATCCC 26  62 323757 Coding NM_000875.2   3301CGGAGATAACTTTTGAGATC 35  63 323758 Coding NM_000875.2   3306GAGACCGGAGATAACTTTTG 29  64 323759 Coding NM_000875.2   3478ATTTTGACTGTGAAATCTTC 13  65 323760 Coding NM_000875.2   3643GCGATCTCCCAGAGGACGAC 72  66 323761 Coding NM_000875.2   3870TGTAGTAGAAGGAGACCTCC 26  67 323762 Coding NM_000875.2   4000GCCTTGTGTCCTGAGTGTCT 84  68 323763 Stop Codon NM_000875.2   4139ATCCAAGGATCAGCAGGTCG 69  69 323764 3′UTR NM_000875.2   4329GCTGCTTGCATATTGAAAAA 77  70 323765 3′UTR NM_000875.2   4334AAAAAGCTGCTTGCATATTG 74  71 323766 3′UTR NM_000875.2   4366GCCCATGTCAGTTAAGGGTT 69  72 323767 3′UTR NM_000875.2   4822CCAGCGTGTCTCTCAAATGG 84  73 323768 Intron NT_035325.2  62268GGAGTTTAAAGGACAGTGCC 59  74 323769 Exon: NT_035325.2 280527CATCACTGACCTCTTTCTAT  0  75 Intron Junction IG-IR 5′ 5′ untranslatedsequence of  76 (M69229) human IGF-IR IGF-IR Nucleotide sequenceencoding  77 (NM0008 human IGF-IR 75) DT1064 Nucleotide sequenceencoding  78 IGF-IR C5 propyne lead CAC AGU UGC UGC AAG DT1064₂  13920antisense oligonucleotide  79 control to human H-ras  18078 antisenseoligonucleotide  80 control to human JNK  15770 antisenseoligonucleotide  81 control to mouse and rat c-raf 161212 PCR primer tohIGF-RI  82 161214 PCR primer to hIGF-RI  83 161215 PCR primer t hIGF-RI 84 129692 Negative control ASO  85 121691 Negative control ASO  86122291 Negative control ASO  87 R451 ASO used for localization study  88251741 ASO used for localization study  89  13920 ASO used forlocalization study  90 147979 ASO used for localization study  91exemplified sense strand  92 exemplified antisense strand  93 PCR primerfor hGAPDH  94 PCR primer for hGAPDH  95 PCR probe to hGAPDH  96 IGF-IRNucleotide Sequence of IGF-IR  97 mRNA with 3′ and 5′ untranslatedregions 391 amino acid sequence of  98 IGF-IR protein 298948 NegativeControl ASO  99 175292 5′UTR SEQ ID NO: 97    930 agtctcaaactcagtcttcg78 100 175293 5′UTR SEQ ID NO: 97     42 gttaatgctggtaaacaaga 40 101175294 5′UTR SEQ ID NO: 97    558 gaagtccgggtcacaggcga 77 102 1752955′UTR SEQ ID NO: 97     29 aacaagagccccagcctcgc 76 103 175296 5′UTR SEQID NO: 97     38 atgctggtaaacaagagccc 57 104 175297 5′UTR SEQ ID NO: 97    37 tgctggtaaacaagagcccc 61 105 175298 5′UTR SEQ ID NO: 97    516ggagtcaaaatgaatgagcg 74 106 175299 5′UTR SEQ ID NO: 97    665aatctgcctaggcgaggaaa 78 107 175300 5′UTR SEQ ID NO: 97     36gctggtaaacaagagcccca 54 108 175301 5′UTR SEQ ID NO: 97    671agcccaaatctgcctaggcg 77 109 175302 5′UTR SEQ ID NO: 97    730cctccattttcaaacccgga 93 110 175303 5′UTR SEQ ID NO: 97    260gaaggtcacagccgaggcga 82 111 175304 5′UTR SEQ ID NO: 97    265tcgctgaaggtcacagccga 76 112 175305 5′UTR SEQ ID NO: 97    410atccaggacacacacaaagc 81 113 175306 5′UTR SEQ ID NO: 97    557aagtccgggtcacaggcgag 54 114 175307 5′UTR SEQ ID NO: 97    931aagtctcaaactcagtcttc 86 115 175308 5′UTR SEQ ID NO: 97    738gtcgtcggcctccattttca 94 116 175309 5′UTR SEQ ID NO: 97    526gcagaaacgcggagtcaaaa 72 117 175310 5′UTR SEQ ID NO: 97    429gcggcgagctccttcccaaa 76 118 175311 5′UTR SEQ ID NO: 97     40taatgctggtaaacaagagc 53 119 175312 5′UTR SEQ ID NO: 97    723tttcaaacccggagaggcag 31 120 175313 5′UTR SEQ ID NO: 97    657taggcgaggaaaaacaagcc 62 121 175314 5′UTR SEQ ID NO: 97    266ctcgctgaaggtcacagccg 87 122 175315 5′UTR SEQ ID NO: 97    798gcagcggcccagggctcggc 75 123 175316 5′UTR SEQ ID NO: 97    267gctcgctgaaggtcacagcc 82 124 175317 5′UTR SEQ ID NO: 97    889cgaaggaaacaatactccga 84 125 175318 5′UTR SEQ ID NO: 97    523gaaacgcggagtcaaaatga 68 126 175319 5′UTR SEQ ID NO: 97    884gaaacaatactccgaagggc 63 127 175320 5′UTR SEQ ID NO: 97    414ccaaatccaggacacacaca 64 128 175321 5′UTR SEQ ID NO: 97    734tcggcctccattttcaaacc 78 129 175322 5′UTR SEQ ID NO: 97    554tccgggtcacaggcgaggcc 67 130 175323 5′UTR SEQ ID NO: 97    508aatgaatgagcggctccccc 82 131 175324 5′UTR SEQ ID NO: 97    261tgaaggtcacagccgaggcg 57 132 175325 5′UTR SEQ ID NO: 97    259aaggtcacagccgaggcgag 55 133 175326 5′UTR SEQ ID NO: 97    415cccaaatccaggacacacac 74 134 175327 5′UTR SEQ ID NO: 97    933acaagtctcaaactcagtct 61 135 175328 5′UTR SEQ ID NO: 97     33ggtaaacaagagccccagcc 64 136  90444    930 cgaagactgagtttgagact 137 90446    558 tcgcctgtgacccggacttc 138  90447     29gcgaggctggggctcttgtt 139  90448     38 gggctcttgtttaccagcat 140  90449    37 ggggctcttgtttaccagca 141  90450    516 cgctcattcattttgactcc 142 90451    665 tttcctcgcctaggcagatt 143  90452     36tggggctcttgtttaccagc 144  90453    671 cgcctaggcagatttgggct 145  90454   730 tccgggtttgaaaatggagg 146  90455    260 tcgcctcggctgtgaccttc 147 90456    265 tcggctgtgaccttcagcga 148  90457    410gctttgtgtgtgtcctggat 149  90458    557 ctcgcctgtgacccggactt 150  90459   931 gaagactgagtttgagactt 151  90460    738 tgaaaatggaggccgacgac 152 90461    526 ttttgactccgcgtttctgc 153  90462    429tttgggaaggagctcgccgc 154  90463     40 gctcttgtttaccagcatta 155  90465   657 ggcttgtttttcctcgccta 156  90466    266 cggctgtgaccttcagcgag 157 90467    798 gccgagccctgggccgctgc 158  90468    267ggctgtgaccttcagcgagc 159  90469    889 tcggagtattgtttccttcg 160  90470   523 tcattttgactccgcgtttc 161  90471    884 gcccttcggagtattgtttc 162 90472    414 tgtgtgtgtcctggatttgg 163  90473    734ggtttgaaaatggaggccga 164  90474    554 ggcctcgcctgtgacccgga 165  90475   508 gggggagccgctcattcatt 166  90476    261 cgcctcggctgtgaccttca 167 90477    259 ctcgcctcggctgtgacctt 168  90478    415gtgtgtgtcctggatttggg 169  90479    933 agactgagtttgagacttgt 170  90480    33 ggctggggctcttgtttacc 171 ¹ASO, antisense oligonucleotide ₂All C'sand U's are C5 propynated

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagrammatic representation of a skin biopsy maintained exvivo.

FIG. 2 is a representation of (A) the nucleotide sequence of the 5′untranslated region of the IGF-IR gene (SEQ ID NO: 76) showing thelocation of targets for ISIS 175314 (SEQ ID NO: 122), ISIS 175317 SEQ IDNO: 125) and ISIS 175323 (SEQ ID NO: 131).

FEATURES Location/Qualifiers source 1..4989 /organism=“Homo sapiens”/db_xref=“taxon:9606” /chromosome=“15” /map=“15q25-q26”/clone=“(lambda)IGF-1-R.85, (lambda)IGF-1-R.76” /tissue_type=“placenta”/clone_lib=“(lamda)gt10” gene 1..4989 /gene=“IGF1R” /note=“synonym:JTK13” /db_xref=“LocusID:3480” /db_xref=“MIM:147370” CDS 46..4149/gene=“IGF1R” /EC_number=“2.7.1.112” /codon_start=1/product=“insulin-like growth factor 1 receptor precursor”/protein_id=“NP_000866.1” /db_xref=“GI:4557665” /db_xref=“LocusID:3480”/db_xref=“MIM:147370” sig_peptide 46..135 /gene=“IGF1R” mat_peptide136..2265 /gene=“IGF1R” /product=“insulin-like growth factor 1 receptoralpha chain” misc_feature 196..531 /gene=“IGF1R” /note=“Recep_L_domain;Region: Receptor L domain. The L domains from these receptors make upthe bilobal ligand binding site. Each L domain consists of asingle-stranded right hand beta-helix. This Pfam entry is missing thefirst 50 amino acid residues of the domain” db_xref=“CDD:pfam01030”misc_feature 568..1044 /gene=“IGF1R” /note=“Furin-like; Region:Furin-like Cysteine rich region” /db_xref=“CDD:pfam00757” misc_feature694..1041 /gene=“IGF1R” /note=“VSP; Region: Giardia variant-specificsurface protein” /db_xref=“CDD:pfam03302” misc_feature 724..855/gene=“IGF1R” /note=“FU; Region: Furin-like repeats”/db_xref=“CDD:smart00261” misc_feature 1168..1479 /gene=“IGF1R”/note=“Recep_L_domain; Region: Receptor L domain. The L domains fromthese receptors make up the bilobal ligand binding site. Each L domainconsists of a single-stranded right hand beta-helix. This Pfam entry ismissing the first 50 amino acid residues of the domain”/db_xref=“CDD:pfam01030” misc_feature 1519..1800 /gene=“IGF1R”/note=“FN3; Region: Fibronectin type 3 domain” /db_xref=“CDD:smart00060”mat_peptide 2266..4146 /gene=“IGF1R” /product=“insulin-like growthfactor 1 receptor beta chain” misc_feature 2542..2787 /gene=“IGF1R”/note=“FN3; Region: Fibronectin type 3 domain” /db_xref=“CDD:smart00060”misc_feature 2548..2796 /gene=“IGF1R” /note=“fn3; Region: Fibronectintype III domain” /db_xref=“CDD:pfam00041” misc_feature 2836..2910/gene=“IGF1R” /note=“transmembrane region (AA 906 - 929);transmembrane-region site” misc_feature 3040..3843 /gene=“IGF1R”/note=“pkinase; Region: Protein kinase domain” /db_xref=“CDD:pfam00069”misc_feature 3040..3843 /gene=“IGF1R” /note=“TyrKc; Region: Tyrosinekinase, catalytic domain” /db_xref=“CDD:smart00219” misc_feature3052..3837 /gene=“IGF1R” /note=“S_TKc; Region: erine/Threonine proteinkinases, catalytic domain” /db_xref=“CDD:smart00220” misc_feature122..2251 /gene=“IGF1R” /note=“alpha-subunit (AA 1 - 710)” misc_feature182..190 /gene=“IGF1R” /note=“pot.N-linked glycosylation site (AA 21 -23)” misc_feature 335..343 /gene=“IGF1R” /note=“pot.N-linkedglycostlation site (AA 72 - 74)” misc_feature 434..442 /gene=“IGF1R”/note=“pot.N-linked glycostlation site (AA 105 - 107)” misc_feature761..769 /gene=“IGF1R” /note=“pot.N-linked glycostlation site (AA 214 -216)” variation 948 /gene=“IGF1R” /allele=“C” /allele=“A”/db_xref=“dbSNP:2229764” misc_feature 971..979 /gene=“IGF1R”/note=“pot.N-linked glycostlation site (AA 284 - 286)” misc_feature1280..1288 /gene=“IGF1R” /note=“pot.N-linked glycostlation site (AA387 - 389)” misc_feature 1343..1351 /gene=“IGF1R” /note=“pot.N-linkedglycosylation site (AA 408 - 410)” misc_feature 1631..1639 /gene=“IGF1R”/note=“pot.N-linked glycostlation site (AA 504 - 506)” variation 1731/gene=“IGF1R” /allele=“G” /allele=“A” /db_xref=“dbSNP:2228531”misc_feature 1850..1858 /gene=“IGF1R” /note=“pot.N-linked glycosylationsite (AA 577 - 579)” misc_feature 1895..1903 /gene=“IGF1R”/note=“pot.N-linked glycosylation site (AA 592 - 594)” misc_feature1949..1957 /gene=“IGF1R” /note=“pot.N-linked glycosylation site (AA610 - 612)” misc_feature 2240..2251 /gene=“IGF1R” /note=“putativeproreceptor processing site (AA 707 - 710)” misc_feature 2252..4132/gene=“IGF1R” /note=“beta-subunit (AA 711 - 1337)” misc_feature2270..2278 /gene=“IGF1R” /note=“pot.N-linked glycosylation site (AA717 - 719]” misc_feature 2297..2305 /gene=“IGF1R” /note=“pot.N-linkedglycosylation site (AA 726 - 728)” misc_feature 2321..2329 /gene=“IGF1R”/note=“pot.N-linked glycosylation site (AA 734 - 736)” variation 2343/gene=“IGF1R” /allele=“T” /allele=“C” /db_xref=“dbSNP:3743262”misc_feature 2729..2737 /gene=“IGF1R” /note=“pot.N-linked glycosylationsite (AA 870 - 872)” misc_feature 2768..2776 /gene=“IGF1R”/note=“pot.N-linked glycosylation site (AA 883 - 885)” misc_feature2918..2926 /gene=“IGF1R” /note=“pot.N-linked glycosylation site (AA933 - 935)” misc_feature 3047..3049 /gene=“IGF1R” /note=“pot.ATP bindingsite (AA 976)” misc_feature 3053..3055 /gene=“IGF1R” /note=“pot.ATPbinding site (AA 978)” misc_feature 3062..3064 /gene=“IGF1R”/note=“pot.ATP binding site (AA 981)” misc_feature 3128..3130/gene=“IGF1R” /note=“pot.ATP binding site (AA 1003)” variation 3174/gene=“IGF1R” /allele=“G” /allele=“A” /db_xref=“dbSNP:2229765” variationcomplement(4205) /allele=“G” /allele=“C” /db xref=“dbSNP:3825954”variation 4267 /gene=“IGF1R” /allele=“T” /allele=“A”/db_xref=“dbSNP:1065304” variation 4268 /gene=“IGF1R” /allele=“T”/allele=“A” /db_xref=“dbSNP:1065305” variation complement(4567)/allele=“AG” /allele=“-” /db_xref=“dbSNP:3833015” BASE COUNT 1216 a 1371c 1320 g 1082 t ORIGIN(B) Nucleotide sequence of IGR-IR and corresponding amino acid sequencewith 3′ and 5′ untranslated regions (NM000).

FIG. 3 is a graphical representation showing (A) the effect of leadIGF-IR ASOs ISIS 175292 through 175328 on IGF-IR mRNA in A549 cellsrelative to negative controls ISIS 13650, ISIS 18078 and ISIS 29848. (B)the effect of lead IGF-IR ASOs ISIS 175314, ISIS 175317 and ISIS 175323on IGF-IR mRNA on A459 cells For (A) & (B), A459 cells were transfectedwith Lipofectin complexed with antisense and control oligonucleotides ata ratio of 2 lipid:1 oligonucleotide. Total cellular RNA was isolated16-20 h later in an automated process (e.g. Qiagen Inc., Valencia,Calif., USA). The histogram represents triplicate measurements from asingle experiment, showing mean IGF-IR mRNA levels as a % of the levelsin untreated control±SD, (C) nucleotide sequences of ASO compounds,control oligonucleotides and primer/probe oligonucleotides.

FIG. 4 is a graphical representation showing the effect of DT1064 (SEQID NO:43) and lead IGF-IR ASOs (ISIS 175314 (SEQ ID NO:27), ISIS 175317(SEQ ID NO:30) and ISIS 175323 (SEQ ID NO:36)) on IGF-IR mRNA levels inHaCaT keratinocytes. 85-90% confluent HaCaT cells were treated with GSV(2 μg/ml), with or without antisense and control oligonucleotides (6.25,25, 100 or 400 nM). Cells were transfected once (18 h before harvest; A)or twice (at 24 and 48 h before harvest; B). Total RNA was recovered andreverse transcribed before being assayed in duplicate by real-time PCR.IGF-IR mRNA was normalized against 18S and expressed as a % of levels inthe GSV-treated control cells. Results represent mean±SEM from duplicatewells of two separate experiments. UT=untreated cells, GSV=cells treatedwith GSV only.

FIG. 5 is a photographic and graphical representation showing the effectof DT1064 (SEQ ID NO:43) and lead IGF-IR ASOs (ISIS 175314 (SEQ IDNO:27), ISIS 175317 (SEQ ID NO:30) and ISIS 175323 (SEQ ID NO:36)) onIGF-IR protein in HaCaT keratinocytes. 85-90% confluent HaCaT cells weretransfected every 24 h for 3 days. Cell lysates were harvested and equalamounts of protein (either 25 or 30 μg) from each sample were resolvedby 7% w/v SDS-PAGE. Protein was transblotted to PVDF membrane and probedwith anti-rabbit IgG recognizing the IGF-IR β subunit. (A) Arepresentative immunoblot (Western 3) showing the intensity of theIGF-IR signal. Samples were run on 4 gels; the GSV-treated and untreatedfrom each gel is shown alongside the samples run on the same gel. (B)Quantitation of IGF-IR protein band intensity expressed as a % of levelsin the GSV-treated control. The histogram shows the mean±SEM for datafrom three separate experiments in which treatments were assessed induplicate. A one-way ANOVA was performed followed by pair-wisecomparisons by Dunnett's test: *P<0.05, ΔP<0.001 versus GSV-treatedcells. UT=untreated cells, GSV=cells treated with GSV only.

FIG. 6 is a graphical representation showing the effect of DT1064 andlead IGF-IR ASOs on cell proliferation rates in HaCaT keratinocytes.Subconfluent HaCaT cells were transfected with GSV alone (2 μg/ml) orGSV (2 μg/ml) complexed with antisense or control oligonucleotides(6.25, 25, 100 or 400 nM) every 24 h for up to 3 days. Cell number wasestimated using amido black assay at the time of the first transfection,and at subsequent 24 h intervals. The data are represented as mean±SEMof two separate experiments in which cell number was determined induplicate. UT=untreated cells, GSV=cells treated with GSV only.

FIG. 7 is a representation of the deoxyribonucleotide sequence of theregion of the IGF-IR gene (M69229; SEQ ID NO:77) showing the location oftargets for ISIS 175314, ISIS 175317 and ISIS 175323.

FIG. 8 is a graphical representation showing the effect of ISIS 175317,IGF-IR lead ASOs, and DT1064 on IGF-I receptor mRNA levels in HaCaTkeratinocytes.

85% confluent HaCaT cells (passage 62-63) were treated for 20 h with GSV(2 μg/ml), with or without antisense or control oligonucleotides (6, 13,25, 50, 100 or 200 nM). Total RNA was extracted and reverse transcribedbefore being assayed in duplicate by real-time PCR. IGF-I receptor mRNAwas normalised against 18S and expressed as a percentage of IGF-IR mRNAlevels in GSV-treated cells. Results represent mean±SD (n=4) fromduplicate wells of two separate experiments. UT=untreated cells,GSV=cells treated with GSV only.

FIG. 9 is a graphical representation showing the effect of ISIS 175317,other IGF-IR lead ASOs, and DT1064 on IGF-I receptor mRNA levels inHaCaT keratinocytes. 85% confluent HaCaT cells (passage 62-63) weretreated for 20 h with GSV (2 μg/ml), with or without antisense orcontrol oligonucleotides (6, 13, 25, 50, 100, or 200 nM). Total RNA wasextracted and reverse transcribed before being assayed in duplicate byreal-time PCR. IGF-I receptor mRNA was normalised against 18S andexpressed as a percentage of IGF-IR mRNA levels in GSV-treated cells.Results represent mean±SD (n=4) from duplicate wells of two separateexperiments. UT=untreated cells, GSV=cells treated with GSV only.

In this experiment Lead IGF-IR ASO: ISIS 175317, Lead IGF-IR ASOs: ISIS323737, ISIS 323744, ISIS 323762, ISIS 323767.

FIG. 10 is a graphical representation show the effect of ISIS 175317,four recently-identified IGF-IR lead ASOs, and DT1064 on IGF-I receptormRNA levels in HaCaT keratinocytes. 85% confluent HaCaT cells (passage45) were treated for 20 h with GSV (2 μg/ml), with or without antisenseor control oligonucleotides (0.3, 1.6, 3, 6, 25, or 100 nM). Total RNAwas extracted and reverse transcribed before being assayed in duplicateby real-time PCR. IGF-I receptor mRNA was normalised against 18S andexpressed as a percentage of the IGF-IR mRNA levels in GSV-treatedcells. Results represent mean±SD (n=2) from duplicate wells of a singleexperiment. UT=untreated cells, GSV=cells treated with GSV only.

In this experiment: Lead IGF-IR ASO: ISIS 175317. Lead IGF-IR ASOs: ISIS323737, ISIS 323744, ISIS 323762, ISIS 323767. Control 2′MOE gapmers:ISIS 129691 (random), ISIS 306064 (8 mismatch). C-5 propyne IGF-IR ASO:DT1064. Control C-5 propyne: 6416 (15 mismatch).

FIG. 11 is a graphical representation showing the concentration-responsecurves for the effects of the four recently identified ASOs and ISIS175317 on relative IGF-IR mRNA levels in HaCaT keratinocytes. 85%confluent HaCaT cells (passage 45) were treated for 20 h with GSV (2μg/ml), with or without antisense or control oligonucleotides (0.4, 1.6,3, 6, 25, or 100 nM). Total RNA was extracted and reverse transcribedbefore being assayed in duplicate by real-time PCR. IGF-I receptor mRNAwas normalised against 18S and expressed as a percentage of GSV-treatedcells. Results represent mean±SD (n=2) from duplicate wells of a singleexperiment. UT=untreated cells, GSV=cells treated with GSV only.

In this experiment: Lead IGF-IR ASO: ISIS 175317. Lead IGF-IR ASOs: ISIS323737, ISIS 323744, ISIS 323762, ISIS 323767.

FIG. 12 is a graphical representation showing mean IGF-IR mRNA levels inpsoriatic skin biopsies after topical application of ISIS 175317 (SEQ IDNO:125). Bars represent the average IGF-IR mRNA level in the epidermisand dermis of vehicle-treated and ISIS 175317 (SEQ ID NO: 125)-treatedbiopsies. Data are expressed relative to the average IGF-IR mRNA levelin the epidermis of the vehicle treated samples, error bars are onestandard deviation. Topically applied ISIS 175317 (SEQ ID NO:125) (10%in ISIS cream) significantly reduced IGF-IR mRNA levels in the epidermisand dermis 24 h after topical application to explants. In all casesn=11. (*=p<0.05, ***=p<0.001).

FIG. 13 is a graphical representation showing mean IGF-IR mRNA levels inpsoriatic skin biopsies after topical application of ISIS 175317 (SEQ IDNO:125). Bars represent the average IGF-IR mRNA level in the psoriaticepidermis and normal epidermis of vehicle-treated and ISIS 175317 (SEQID NO:125)-treated biopsies. Data are expressed relative to the averageIGF-IR mRNA level in the epidermis of the vehicle treated samples, errorbars are one standard deviation. Topically applied ISIS 175317 (SEQ IDNO:125) (10% in ISIS cream) significantly reduced IGF-IR mRNA levels inthe epidermis and dermis 24 h after topical application to explants. Inall cases n=11. (*=p<0.05, ***=p<0.001).

FIG. 14 is a graphical representation showing mean IGF-IR mRNA levels inpsoriatic skin biopsies after topical application of ISIS 175317 toepidermis tissue showing specificity of ISIS 175317 to IGF-IR and not toGAPDH, HPRT, insulin receptor, Casp. 3 and Bax. Bars represent theaverage IGF-IR mRNA level in the epidermis and dermis of vehicle-treatedand ISIS 175317-treated biopsies. Data are expressed relative to theaverage IGF-IR mRNA level in the epidermis of the vehicle treatedsamples, error bars are one standard deviation. Topically applied ISIS175317 (10% in ISIS cream) significantly reduced IGF-IR mRNA levels inthe epidermis and dermis 24 h after topical application to explants. Inall cases n=11. (*=p<0.05, ***=p<0.001).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

A. Overview of the Invention

The present invention employs compounds, preferably oligonucleotides andsimilar species for use in modulating the function or effect of nucleicacid molecules encoding the Insulin Like Growth Factor I receptor and,in a particular embodiment, the human Insulin Like Growth Factor-Ireceptor (IGF-IR). This is accomplished by providing oligonucleotideswhich specifically hybridize with one or more nucleic acid moleculesencoding IGF-IR. As used herein, the terms “target nucleic acid” and“nucleic acid molecule encoding IGF-IR” have been used for convenienceto encompass DNA encoding IGF-IR, RNA (including pre-mRNA and mRNA orportions thereof) transcribed from such DNA, and also cDNA derived fromsuch RNA. The hybridization of a compound of this invention with itstarget nucleic acid is generally referred to as “antisense”.Consequently, the preferred mechanism believed to be included in thepractice of some preferred embodiments of the invention is referred toherein as “antisense inhibition.” Such antisense inhibition is typicallybased upon hydrogen bonding-based hybridization of oligonucleotidestrands or segments such that at least one strand or segment is cleaved,degraded, or otherwise rendered inoperable. In this regard, it ispresently preferred to target specific nucleic acid molecules and theirfunctions for such antisense inhibition.

The functions of DNA to be interfered with can include replication andtranscription. Replication and transcription, for example, can be froman endogenous cellular template, a vector, a plasmid construct orotherwise. The functions of RNA to be interfered with can includefunctions such as translocation of the RNA to a site of proteintranslation, translocation of the RNA to sites within the cell which aredistant from the site of RNA synthesis, translation of protein from theRNA, splicing of the RNA to yield one or more RNA species, and catalyticactivity or complex formation involving the RNA which may be engaged inor facilitated by the RNA. One preferred result of such interferencewith target nucleic acid function is modulation of the expression ofIGF-1R. In the context of the present invention, “modulation” and“modulation of expression” mean either an increase (stimulation) or adecrease (inhibition) in the amount or levels of a nucleic acid moleculeencoding the gene, e.g., DNA or RNA. Inhibition is often the preferredform of modulation of expression and mRNA is often a preferred targetnucleic acid.

In the context of this invention, “hybridization” means the pairing ofcomplementary strands of oligomeric compounds. In the present invention,the preferred mechanism of pairing involves hydrogen bonding, which maybe Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding,between complementary nucleoside or nucleotide bases (nucleobases) ofthe strands of oligomeric compounds. For example, adenine and thymineare complementary nucleobases which pair through the formation ofhydrogen bonds. Hybridization can occur under varying circumstances.

An antisense compound is specifically hybridizable when binding of thecompound to the target nucleic acid interferes with the normal functionof the target nucleic acid to cause a loss of activity, and there is asufficient degree of complementarity to avoid non-specific binding ofthe antisense compound to non-target nucleic acid sequences underconditions in which specific binding is desired, i.e., underphysiological conditions in the case of in vivo assays or therapeutictreatment, and under conditions in which assays are performed in thecase of in vitro assays.

In the present invention the phrase “stringent hybridization conditions”or “stringent conditions” refers to conditions under which a compound ofthe invention will hybridize to its target sequence, but to a minimalnumber of other sequences. Stringent conditions are sequence-dependentand will be different in different circumstances and in the context ofthis invention, “stringent conditions” under which oligomeric compoundshybridize to a target sequence are determined by the nature andcomposition of the oligomeric compounds and the assays in which they arebeing investigated.

“Complementary,” as used herein, refers to the capacity for precisepairing between two nucleobases of an oligomeric compound. For example,if a nucleobase at a certain position of an oligonucleotide (anoligomeric compound), is capable of hydrogen bonding with a nucleobaseat a certain position of a target nucleic acid, said target nucleic acidbeing a DNA, RNA, or oligonucleotide molecule, then the position ofhydrogen bonding between the oligonucleotide and the target nucleic acidis considered to be a complementary position. The oligonucleotide andthe further DNA, RNA, or oligonucleotide molecule are complementary toeach other when a sufficient number of complementary positions in eachmolecule are occupied by nucleobases which can hydrogen bond with eachother. Thus, “specifically hybridizable” and “complementary” are termswhich are used to indicate a sufficient degree of precise pairing orcomplementarity over a sufficient number of nucleobases such that stableand specific binding occurs between the oligonucleotide and a targetnucleic acid.

It is understood in the art that the sequence of an antisense compoundneed not be 100% complementary to that of its target nucleic acid to bespecifically hybridizable. Moreover, an oligonucleotide may hybridizeover one or more segments such that intervening or adjacent segments arenot involved in the hybridization event (e.g., a loop structure orhairpin structure). It is preferred that the antisense compounds of thepresent invention comprise at least 70% sequence complementarity to atarget region within the target nucleic acid, more preferably that theycomprise 90% sequence complementarity and even more preferably comprise95% sequence complementarity to the target region within the targetnucleic acid sequence to which they are targeted. For example, anantisense compound in which 18 of 20 nucleobases of the antisensecompound are complementary to a target region, and would thereforespecifically hybridize, would represent 90 percent complementarity. Inthis example, the remaining noncomplementary nucleobases may beclustered or interspersed with complementary nucleobases and need not becontiguous to each other or to complementary nucleobases. As such, anantisense compound which is 18 nucleobases in length having 4 (four)noncomplementary nucleobases which are flanked by two regions ofcomplete complementarity with the target nucleic acid would have 77.8%overall complementarity with the target nucleic acid and would thus fallwithin the scope of the present invention. Percent complementarity of anantisense compound with a region of a target nucleic acid can bedetermined routinely using BLAST programs (basic local alignment searchtools) and PowerBLAST programs known in the art (Altschul et al., J.Mol. Biol. 215: 403-410, 1990; Zhang and Madden, Genome Res. 7: 649-656,1997).

B. Compounds of the Invention

According to the present invention, compounds include antisenseoligomeric compounds, antisense oligonucleotides, ribozymes, externalguide sequence (EGS) oligonucleotides, alternate splicers, primers,probes, and other oligomeric compounds which hybridize to at least aportion of the target nucleic acid. As such, these compounds may beintroduced in the form of single-stranded, double-stranded, circular orhairpin oligomeric compounds and may contain structural elements such asinternal or terminal bulges or loops. Once introduced to a system, thecompounds of the invention may elicit the action of one or more enzymesor structural proteins to effect modification of the target nucleicacid.

One non-limiting example of such an enzyme is RNAse H, a cellularendonuclease which cleaves the RNA strand of an RNA:DNA duplex. It isknown in the art that single-stranded antisense compounds which are“DNA-like” elicit RNAse H. Activation of RNase H, therefore, results incleavage of the RNA target, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. Similar roleshave been postulated for other ribonucleases such as those in the RNaseIII and ribonuclease L family of enzymes.

While the preferred form of antisense compound is a single-strandedantisense oligonucleotide, in many species the introduction ofdouble-stranded structures, such as double-stranded RNA (dsRNA)molecules, has been shown to induce potent and specificantisense-mediated reduction of the function of a gene or its associatedgene products. This phenomenon occurs in both plants and animals and isbelieved to have an evolutionary connection to viral defense andtransposon silencing.

The first evidence that dsRNA could lead to gene silencing in animalscame in 1995 from work in the nematode, Caenorhabditis elegans (Guo andKempheus, Cell 81: 611-620, 1995). Montgomery et al. have shown that theprimary interference effects of dsRNA are posttranscriptional(Montgomery et al., Proc. Natl. Acad. Sci. USA. 95: 15502-15507, 1998).The post-transcriptional antisense mechanism defined in Caenorhabditiselegans resulting from exposure to double-stranded RNA (dsRNA) has sincebeen designated RNA interference (RNAi). This term has been generalizedto mean antisense-mediated gene silencing involving the introduction ofdsRNA leading to the sequence-specific reduction of endogenous targetedmRNA levels (Fire et al., Nature 391: 806-811, 1998). Recently, it hasbeen shown that it is, in fact, the single-stranded RNA oligomers ofantisense polarity of the dsRNAs which are the potent inducers of RNAi(Tijsterman et al., Science, 295; 694-697, 2002).

In the context of this invention, the term “oligomeric compound” refersto a polymer or oligomer comprising a plurality of monomeric units. Inthe context of this invention, the term “oligonucleotide” refers to anoligomer or polymer of ribonucleic acid (RNA) or deoxyribonucleic acid(DNA) or mimetics, chimeras, analogs and homologs thereof. This termincludes oligonucleotides composed of naturally occurring nucleobases,sugars and covalent internucleoside (backbone) linkages as well asoligonucleotides having non-naturally occurring portions which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of desirable properties such as, forexample, enhanced cellular uptake, enhanced affinity for a targetnucleic acid and increased stability in the presence of nucleases.

While oligonucleotides are a preferred form of the compounds of thisinvention, the present invention comprehends other families of compoundsas well, including but not limited to oligonucleotide analogs andmimetics such as those described herein.

The compounds in accordance with this invention preferably comprise fromabout 8 to about 80 nucleobases (i.e. from about 8 to about 80 linkednucleosides). One of ordinary skill in the art will appreciate that theinvention embodies compounds of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, or 80 nucleobases in length.

In one preferred embodiment, the compounds of the invention are 12 to 50nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleobases inlength.

In another preferred embodiment, the compounds of the invention are 15to 30 nucleobases in length. One having ordinary skill in the art willappreciate that this embodies compounds of 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length.

Particularly preferred compounds are oligonucleotides from about 12 toabout 50 nucleobases, even more preferably those comprising from about15 to about 30 nucleobases.

Antisense compounds 8-80 nucleobases in length comprising a stretch ofat least eight (8) consecutive nucleobases selected from within theillustrative antisense compounds are considered to be suitable antisensecompounds as well.

Exemplary preferred antisense compounds include oligonucleotidesequences that comprise at least the 8 consecutive nucleobases from the5′-terminus of one of the illustrative preferred antisense compounds(the remaining nucleobases being a consecutive stretch of the sameoligonucleotide beginning immediately upstream of the 5′-terminus of theantisense compound which is specifically hybridizable to the targetnucleic acid and continuing until the oligonucleotide contains about 8to about 80 nucleobases). Similarly preferred antisense compounds arerepresented by oligonucleotide sequences that comprise at least the 8consecutive nucleobases from the 3′-terminus of one of the illustrativepreferred antisense compounds (the remaining nucleobases being aconsecutive stretch of the same oligonucleotide beginning immediatelydownstream of the 3′-terminus of the antisense compound which isspecifically hybridizable to the target nucleic acid and continuinguntil the oligonucleotide contains about 8 to about 80 nucleobases). Onehaving skill in the art armed with the preferred antisense compoundsillustrated herein will be able, without undue experimentation, toidentify further preferred antisense compounds.

The candidate compounds of the present invention are referred to hereinby ISIS number or SEQ ID NO. Preferred compounds are shown in Table 1.

Candidate compounds are also referred to herein as “lead” compounds.

One group of particularly preferred ASOs include ISIS 175308 (SEQ ID NO:116), ISIS 175302 (SEQ ID NO: 110), ISIS 175314 (SEQ ID NO: 122), ISIS175307 (SEQ ID NO: 115), ISIS 175317 (SEQ ID NO: 125) and ISIS 175323(SEQ ID NO: 131).

Another group of particularly preferred ASOs include ISIS 323744 (SEQ IDNO:50), ISIS 323747 (SEQ ID NO:53), ISIS 323767 (SEQ ID NO:73), ISIS323762 (SEQ ID NO:68) and ISIS 323737 (SEQ ID NO:43).

An even more particularly preferred ASO is ISIS 175317 (SEQ ID NO: 125).

C. Targets of the Invention

“Targeting” an antisense compound to a particular nucleic acid molecule,in the context of this invention, can be a multistep process. Theprocess usually begins with the identification of a target nucleic acidwhose function is to be modulated. This target nucleic acid may be, forexample, a cellular gene (or mRNA transcribed from the gene) whoseexpression is associated with a particular disorder or disease state, ora nucleic acid molecule from an infectious agent. In the presentinvention, the target nucleic acid encodes IGF-IR.

The targeting process usually also includes determination of at leastone target region, segment, or site within the target nucleic acid forthe antisense interaction to occur such that the desired effect, e.g.,modulation of expression, will result. Within the context of the presentinvention, the term “region” is defined as a portion of the targetnucleic acid having at least one identifiable structure, function, orcharacteristic. Within regions of target nucleic acids are segments.“Segments” are defined as smaller or sub-portions of regions within atarget nucleic acid. “Sites,” as used in the present invention, aredefined as positions within a target nucleic acid.

Since, as is known in the art, the translation initiation codon istypically 5′-AUG (in transcribed mRNA molecules; 5′-ATG in thecorresponding DNA molecule), the translation initiation codon is alsoreferred to as the “AUG codon,” the “start codon” or the “AUG startcodon”. A minority of genes have a translation initiation codon havingthe RNA sequence 5′-GUG, 5′-UUG or 5′-CUG, and 5′-AUA, 5′-ACG and 5′-CUGhave been shown to function in vivo. Thus, the terms “translationinitiation codon” and “start codon” can encompass many codon sequences,even though the initiator amino acid in each instance is typicallymethionine (in eukaryotes) or formylmethionine (in prokaryotes). It isalso known in the art that eukaryotic and prokaryotic genes may have twoor more alternative start codons, any one of which may be preferentiallyutilized for translation initiation in a particular cell type or tissue,or under a particular set of conditions. In the context of theinvention, “start codon” and “translation initiation codon” refer to thecodon or codons that are used in vivo to initiate translation of an mRNAtranscribed from a gene encoding IGF-IR, regardless of the sequence(s)of such codons. It is also known in the art that a translationtermination codon (or “stop codon”) of a gene may have one of threesequences, i.e., 5′-UAA, 5′-UAG and 5′-UGA (the corresponding DNAsequences are 5′-TAA, 5′-TAG and 5′-TGA, respectively).

The terms “start codon region” and “translation initiation codon region”refer to a portion of such an mRNA or gene that encompasses from about25 to about 50 contiguous nucleotides in either direction (i.e., 5′ or3′) from a translation initiation codon. Similarly, the terms “stopcodon region” and “translation termination codon region” refer to aportion of such an mRNA or gene that encompasses from about 25 to about50 contiguous nucleotides in either direction (i.e., 5′ or 3′) from atranslation termination codon. Consequently, the “start codon region”(or “translation initiation codon region”) and the “stop codon region”(or “translation termination codon region”) are all regions which may betargeted effectively with the antisense compounds of the presentinvention.

The open reading frame (ORF) or “coding region,” which is known in theart to refer to the region between the translation initiation codon andthe translation termination codon, is also a region which may betargeted effectively. Within the context of the present invention, apreferred region is the intragenic region encompassing the translationinitiation or termination codon of the open reading frame (ORF) of agene.

Other target regions include the 5′ untranslated region (5′UTR), knownin the art to refer to the portion of an mRNA in the 5′ direction fromthe translation initiation codon, and thus including nucleotides betweenthe 5′ cap site and the translation initiation codon of an mRNA (orcorresponding nucleotides on the gene), and the 3′ untranslated region(3′UTR), known in the art to refer to the portion of an mRNA in the 3′direction from the translation termination codon, and thus includingnucleotides between the translation termination codon and 3′ end of anmRNA (or corresponding nucleotides on the gene). The 5′ cap site of anmRNA comprises an N7-methylated guanosine residue joined to the 5′-mostresidue of the mRNA via a 5′-5′ triphosphate linkage. The 5′ cap regionof an mRNA is considered to include the 5′ cap structure itself as wellas the first 50 nucleotides adjacent to the cap site. It is alsopreferred to target the 5′ cap region.

Although some eukaryotic mRNA transcripts are directly translated, manycontain one or more regions, known as “introns,” which are excised froma transcript before it is translated. The remaining (and thereforetranslated) regions are known as “exons” and are spliced together toform a continuous mRNA sequence. Targeting splice sites, i.e.,intron-exon junctions or exon-intron junctions, may also be particularlyuseful in situations where aberrant splicing is implicated in disease,or where an overproduction of a particular splice product is implicatedin disease. Aberrant fusion junctions due to rearrangements or deletionsare also preferred target sites. mRNA transcripts produced via theprocess of splicing of two (or more) mRNAs from different gene sourcesare known as “fusion transcripts”. It is also known that introns can beeffectively targeted using antisense compounds targeted to, for example,DNA or pre-mRNA.

It is also known in the art that alternative RNA transcripts can beproduced from the same genomic region of DNA. These alternativetranscripts are generally known as “variants”. More specifically,“pre-mRNA variants” are transcripts produced from the same genomic DNAthat differ from other transcripts produced from the same genomic DNA ineither their start or stop position and contain both intronic and exonicsequence.

Upon excision of one or more exon or intron regions, or portions thereofduring splicing, pre-mRNA variants produce smaller “mRNA variants”.Consequently, mRNA variants are processed pre-mRNA variants and eachunique pre-mRNA variant must always produce a unique mRNA variant as aresult of splicing. These mRNA variants are also known as “alternativesplice variants”. If no splicing of the pre-mRNA variant occurs then thepre-mRNA variant is identical to the mRNA variant.

It is also known in the art that variants can be produced through theuse of alternative signals to start or stop transcription and thatpre-mRNAs and mRNAs can possess more that one start codon or stop codon.Variants that originate from a pre-mRNA or mRNA that use alternativestart codons are known as “alternative start variants” of that pre-mRNAor mRNA. Those transcripts that use an alternative stop codon are knownas “alternative stop variants” of that pre-mRNA or mRNA. One specifictype of alternative stop variant is the “polyA variant” in which themultiple transcripts produced result from the alternative selection ofone of the “polyA stop signals” by the transcription machinery, therebyproducing transcripts that terminate at unique polyA sites. Within thecontext of the invention, the types of variants described herein arealso preferred target nucleic acids.

The locations on the target nucleic acid to which the preferredantisense compounds hybridize are hereinbelow referred to as “preferredtarget segments.” As used herein the term “preferred target segment” isdefined as at least an 8-nucleobase portion of a target region to whichan active antisense compound is targeted. While not wishing to be boundby theory, it is presently believed that these target segments representportions of the target nucleic acid which are accessible forhybridization.

While the specific sequences of certain preferred target segments areset forth herein, one of skill in the art will recognize that theseserve to illustrate and describe particular embodiments within the scopeof the present invention. Additional preferred target segments may beidentified by one having ordinary skill.

Target segments 8-80 nucleobases in length comprising a stretch of atleast eight (8) consecutive nucleobases selected from within theillustrative preferred target segments are considered to be suitable fortargeting as well.

Target segments can include DNA or RNA sequences that comprise at leastthe 8 consecutive nucleobases from the 5′-terminus of one of theillustrative preferred target segments (the remaining nucleobases beinga consecutive stretch of the same DNA or RNA beginning immediatelyupstream of the 5′-terminus of the target segment and continuing untilthe DNA or RNA contains about 8 to about 80 nucleobases). Similarlypreferred target segments are represented by DNA or RNA sequences thatcomprise at least the 8 consecutive nucleobases from the 3′-terminus ofone of the illustrative preferred target segments (the remainingnucleobases being a consecutive stretch of the same DNA or RNA beginningimmediately downstream of the 3′-terminus of the target segment andcontinuing until the DNA or RNA contains about 8 to about 80nucleobases). One having skill in the art armed with the preferredtarget segments illustrated herein will be able, without undueexperimentation, to identify further preferred target segments.

Once one or more target regions, segments or sites have been identified,antisense compounds are chosen which are sufficiently complementary tothe target, i.e., hybridize sufficiently well and with sufficientspecificity, to give the desired effect.

D. Screening and Target Validation

In a further embodiment, the “preferred target segments” identifiedherein may be employed in a screen for additional compounds thatmodulate the expression of the IGF-IR gene. “Modulators” are thosecompounds that decrease or increase the expression of a nucleic acidmolecule encoding IGF-IR and which comprise at least a 8-nucleobaseportion which is complementary to a preferred target segment. Thescreening method comprises the steps of contacting a preferred targetsegment of a nucleic acid molecule encoding IGF-IR with one or morecandidate modulators, and selecting for one or more candidate modulatorswhich decrease or increase the expression of a nucleic acid moleculeencoding IGF-IR. Once it is shown that the candidate modulator ormodulators are capable of modulating (e.g. either decreasing orincreasing) the expression of a nucleic acid molecule encoding IGF-IR,the modulator may then be employed in further investigative studies ofthe function of IGF-1R, or for use as a research, diagnostic, ortherapeutic agent in accordance with the present invention.

The preferred target segments of the present invention may be also becombined with their respective complementary antisense compounds of thepresent invention to form stabilized double-stranded (duplexed)oligonucleotides.

Such double stranded oligonucleotide moieties have been shown in the artto modulate target expression and regulate translation as well as RNAprocessing via an antisense mechanism. Moreover, the double-strandedmoieties may be subject to chemical modifications (Fire et al., Nature391: 806-811, 1998; Timmons and Fire, Nature 395: 854, 1998; Timmons etal., Gene 263: 103-112, 2001; Tabara et al., Science 282: 430-431, 1998;Montgomery et al., 1998, supra; Tuschl et al., Genes Dev. 13: 3191-3197,1999; Elbashir et al., Nature, 411: 494-498, 2001; Elbashir et al.,Genes Dev. 15: 188-200, 2001). For example, such double-strandedmoieties have been shown to inhibit the target by the classicalhybridization of antisense strand of the duplex to the target, therebytriggering enzymatic degradation of the target (Tijsterman et al., 2002,supra).

The compounds of the present invention can also be applied in the areasof drug discovery and target validation. The present inventioncomprehends the use of the compounds and preferred target segmentsidentified herein in drug discovery efforts to elucidate relationshipsthat exist between IGF-I, IGF-IR or IGF-I/IGF-IR interaction and adisease state, phenotype, or condition. These methods include detectingor modulating IGF-IR comprising contacting a sample, tissue, cell, ororganism with the compounds of the present invention, measuring thenucleic acid or protein level of IGF-IR and/or a related phenotypic orchemical endpoint at some time after treatment, and optionally comparingthe measured value to a non-treated sample or sample treated with afurther compound of the invention. These methods can also be performedin parallel or in combination with other experiments to determine thefunction of unknown genes for the process of target validation or todetermine the validity of a particular gene product as a target fortreatment or prevention of a particular disease, condition, orphenotype.

E. Kits, Research Reagents, Diagnostics, and Therapeutics

The compounds of the present invention can be utilized for diagnostics,therapeutics, prophylaxis and as research reagents and kits.Furthermore, antisense oligonucleotides, which are able to inhibit geneexpression with exquisite specificity, are often used by those ofordinary skill to elucidate the function of particular genes or todistinguish between functions of various members of a biologicalpathway.

For use in kits and diagnostics, the compounds of the present invention,either alone or in combination with other compounds or therapeutics, canbe used as tools in differential and/or combinatorial analyses toelucidate expression patterns of a portion or the entire complement ofgenes expressed within cells and tissues.

As one non-limiting example, expression patterns within cells or tissuestreated with one or more antisense compounds are compared to controlcells or tissues not treated with antisense compounds and the patternsproduced are analyzed for differential levels of gene expression as theypertain, for example, to disease association, signaling pathway,cellular localization, expression level, size, structure or function ofthe genes examined. These analyses can be performed on stimulated orunstimulated cells and in the presence or absence of other compoundswhich affect expression patterns.

Examples of methods of gene expression analysis known in the art includeDNA arrays or microarrays (Brazma and Vilo, FEBS Lett. 480: 17-24, 2000;Celis et al., FEBS Lett. 480: 2-16, 2000), SAGE (serial analysis of geneexpression)(Madden et al., Drug Discov. Today 5: 415-425, 2000), READS(restriction enzyme amplification of digested cDNAs) (Prashar andWeissman, Methods Enzymol. 303: 258-272, 1999), TOGA (total geneexpression analysis) (Sutcliffe et al., Proc. Natl. Acad. Sci. USA 97:1976-1981, 2000), protein arrays and proteomics (Celis et al. 2000,supra; Jungblut et al., Electrophoresis 20: 2100-2110, 1999), expressedsequence tag (EST) sequencing (Celis et al., 2000, supra; Larsson etal., J. Biotechnol. 80: 143-157, 2000), subtractive RNA fingerprinting(SuRF) (Fuchs et al., Anal. Biochem. 286: 91-98, 2000; Larson et al.,Cytometry 41: 203-208, 2000), subtractive cloning, differential display(DD) (Jurecic and Belmont, Curr. Opin. Microbiol. 3: 316-321, 2000),comparative genomic hybridization (Carulli et al., J. Cell Biochem.Suppl. 31: 286-296, 1998), FISH (fluorescent in situ hybridization)techniques (Going and Gusterson, Eur. J. Cancer, 35: 1895-1904, 1999)and mass spectrometry methods (To, Comb. Chem. High Throughput Screen,3: 235-241, 2000).

The compounds of the invention are useful for research and diagnostics,because these compounds hybridize to nucleic acids encoding IGF-IR. Forexample, oligonucleotides that are shown to hybridize with suchefficiency and under such conditions as disclosed herein as to beeffective IGF-IR inhibitors of IGF-IR gene expression inhibitors willalso be effective primers or probes under conditions favoring geneamplification or detection, respectively. These primers and probes areuseful in methods requiring the specific detection of nucleic acidmolecules encoding IGF-IR and in the amplification of said nucleic acidmolecules for detection or for use in further studies of IGF-IR or itsgene. Hybridization of the antisense oligonucleotides, particularly theprimers and probes, of the invention with a nucleic acid encoding IGF-IRcan be detected by means known in the art. Such means may includeconjugation of an enzyme to the oligonucleotide, radiolabelling of theoligonucleotide or any other suitable detection means. Kits using suchdetection means for detecting the level of IGF-IR in a sample may alsobe prepared.

The specificity and sensitivity of antisense is also harnessed by thoseof skill in the art for therapeutic uses. Antisense compounds have beenemployed as therapeutic moieties in the treatment of disease states inanimals, including humans. Antisense oligonucleotide drugs, includingribozymes, have been safely and effectively administered to humans andnumerous clinical trials are presently underway. It is thus establishedthat antisense compounds can be useful therapeutic modalities that canbe configured to be useful in treatment regimes for the treatment ofcells, tissues and animals, especially humans.

For therapeutics, an animal, preferably a human, suspected of having adisease or disorder which can be treated by modulating the expression ofthe IGF-IR gene is treated by administering antisense compounds inaccordance with this invention. For example, in one non-limitingembodiment, the methods comprise the step of administering to the animalin need of treatment, a therapeutically effective amount of an IGF-IRgene expression inhibitor. The IGF-IR gene expression inhibitors of thepresent invention effectively inhibit the activity of the IGF-IR proteinor inhibit the expression of the IGF-IR gene. In one embodiment, theactivity or expression of IGF-IR or its gene in an animal is inhibitedby about 10%. Preferably, the activity or expression of IGF-IR or itsgene in an animal is inhibited by about 30%. More preferably, theactivity or expression of IGF-IR or its gene in an animal is inhibitedby 50% or more.

For example, the reduction of the expression of the IGF-IR gene may bemeasured in serum, adipose tissue, skin cells, liver or any other bodyfluid, tissue or organ of the animal. Preferably, the cells containedwithin said fluids, tissues or organs being analyzed contain a nucleicacid molecule encoding an IGF-IR protein.

The compounds of the invention can be utilized in pharmaceuticalcompositions by adding an effective amount of a compound to a suitablepharmaceutically acceptable diluent or carrier. Use of the compounds andmethods of the invention may also be useful prophylactically.

F. Modifications

As is known in the art, a nucleoside is a base-sugar combination. Thebase portion of the nucleoside is normally a heterocyclic base. The twomost common classes of such heterocyclic bases are the purines and thepyrimidines. Nucleotides are nucleosides that further include aphosphate group covalently linked to the sugar portion of thenucleoside. For those nucleosides that include a pentofuranosyl sugar,the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxylmoiety of the sugar. In forming oligonucleotides, the phosphate groupscovalently link adjacent nucleosides to one another to form a linearpolymeric compound. In turn, the respective ends of this linearpolymeric compound can be further joined to form a circular compound,however, linear compounds are generally preferred. In addition, linearcompounds may have internal nucleobase complementarity and may thereforefold in a manner as to produce a fully or partially double-strandedcompound. Within oligonucleotides, the phosphate groups are commonlyreferred to as forming the internucleoside backbone of theoligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′to 5′ phosphodiester linkage.

Modified Internucleoside Linkages (Backbones)

Specific examples of preferred antisense compounds useful in thisinvention include oligonucleotides containing modified backbones ornon-natural internucleoside linkages. As defined in this specification,oligonucleotides having modified backbones include those that retain aphosphorus atom in the backbone and those that do not have a phosphorusatom in the backbone. For the purposes of this specification, and assometimes referenced in the art, modified oligonucleotides that do nothave a phosphorus atom in their internucleoside backbone can also beconsidered to be oligonucleosides.

Preferred modified oligonucleotide backbones containing a phosphorusatom therein include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiralphosphonates, phosphinates, phosphoramidates including 3′-aminophosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, selenophosphatesand boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogsof these, and those having inverted polarity wherein one or moreinternucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage.Preferred oligonucleotides having inverted polarity comprise a single 3′to 3′ linkage at the 3′-most internucleotide linkage i.e. a singleinverted nucleoside residue which may be abasic (the nucleobase ismissing or has a hydroxyl group in place thereof). Various salts, mixedsalts and free acid forms are also included.

Representative United States patents that teach the preparation of theabove phosphorus-containing linkages include, but are not limited to,U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and5,625,050, certain of which are commonly owned with this application,and each of which is herein incorporated by reference.

Preferred modified oligonucleotide backbones that do not include aphosphorus atom therein have backbones that are formed by short chainalkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkylor cycloalkyl internucleoside linkages, or one or more short chainheteroatomic or heterocyclic internucleoside linkages. These includethose having morpholino linkages (formed in part from the sugar portionof a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; riboacetyl backbones; alkene containingbackbones; sulfamate backbones; methyleneimino and methylenehydrazinobackbones; sulfonate and sulfonamide backbones; amide backbones; andothers having mixed N, O, S and CH₂ component parts.

Representative United States patents that teach the preparation of theabove oligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312;5,633,360; 5,677,437; 5,792,608; 5,646,269 and 5,677,439, certain ofwhich are commonly owned with this application, and each of which isherein incorporated by reference.

Modified Sugar and Internucleoside Linkages-Mimetics

In other preferred oligonucleotide mimetics, both the sugar and theinternucleoside linkage (i.e. the backbone), of the nucleotide units arereplaced with novel groups. The nucleobase units are maintained forhybridization with an appropriate target nucleic acid. One suchcompound, an oligonucleotide mimetic that has been shown to haveexcellent hybridization properties, is referred to as a peptide nucleicacid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotideis replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative United States patents that teach thepreparation of PNA compounds include, but are not limited to, U.S. Pat.Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is hereinincorporated by reference. Further teaching of PNA compounds can befound in Nielsen et al., Science 254: 1497-1500, 1991.

Preferred embodiments of the invention are oligonucleotides withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—O—CH₂—, —CH₂—N(CH₃)—O—CH₂— [knownas a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —O—N(CH₃)—CH₂—CH₂— [wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of the abovereferenced U.S. Pat. No. 5,489,677, and the amide backbones of the abovereferenced U.S. Pat. No. 5,602,240. Also preferred are oligonucleotideshaving morpholino backbone structures of the above-referenced U.S. Pat.No. 5,034,506.

Modified Sugars

Modified oligonucleotides may also contain one or more substituted sugarmoieties. Preferred oligonucleotides comprise one of the following atthe 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—,S—or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀alkenyl and alkynyl. Particularly preferred are O[(CH₂)_(n)O]_(m)CH₃,O(CH₂)_(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, andO(CH₂)_(n)ON[(CH₂)_(n)CH₃]₂, where n and m are from 1 to about 10. Otherpreferred oligonucleotides comprise one of the following at the 2′position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkenyl,alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl,Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl,heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl,an RNA cleaving group, a reporter group, an intercalator, a group forimproving the pharmacokinetic properties of an oligonucleotide, or agroup for improving the pharmacodynamic properties of anoligonucleotide, and other substituents having similar properties. Apreferred modification includes 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, alsoknown as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chim.Acta, 78: 486-504, 1995) i.e., an alkoxyalkoxy group. A furtherpreferred modification includes 2′-dimethylaminooxyethoxy, i.e., aO(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in exampleshereinbelow, and 2′-dimethylamino-ethoxyethoxy (also known in the art as2′-O-dimethyl-amino-ethoxy-ethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₃)₂, also described in examples hereinbelow.

Other preferred modifications include 2′-methoxy (2′-O—CH₃),2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂), 2′-allyl (2′-CH₂—CH═CH₂), 2′-O-allyl(2′-O—CH₂—CH═CH₂) and 2′-fluoro (2′-F). The 2′-modification may be inthe arabino (up) position or ribo (down) position. A preferred2′-arabino modification is 2′-F. Similar modifications may also be madeat other positions on the oligonucleotide, particularly the 3′ positionof the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide.Oligonucleotides may also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative UnitedStates patents that teach the preparation of such modified sugarstructures include, but are not limited to, U.S. Pat. Nos. 4,981,957;5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909;5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633;5,792,747; and 5,700,920, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference in its entirety.

A further preferred modification of the sugar includes Locked NucleicAcids (LNAs) in which the 2′-hydroxyl group is linked to the 3′ or 4′carbon atom of the sugar ring, thereby forming a bicyclic sugar moiety.The linkage is preferably a methylene (—CH₂—)_(n) group bridging the 2′oxygen atom and the 4′ carbon atom wherein n is 1 or 2. LNAs andpreparation thereof are described in WO 98/39352 and WO 99/14226.

Natural and Modified Nucleobases

Oligonucleotides may also include nucleobase (often referred to in theart simply as “base”) modifications or substitutions. As used herein,“unmodified” or “natural” nucleobases include the purine bases adenine(A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C)and uracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl (—C≡C—CH₃) uracil and cytosine andother alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosineand thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino,8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified nucleobases include tricyclicpyrimidines such as phenoxazine cytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps such as asubstituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobasesmay also include those in which the purine or pyrimidine base isreplaced with other heterocycles, for example 7-deaza-adenine,7-deazaguanosine, 2-aminopyridine and 2-pyridone. Further nucleobasesinclude those disclosed in U.S. Pat. No. 3,687,808, those disclosed inThe Concise Encyclopedia Of Polymer Science And Engineering, pages858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosedby Englisch et al., Angewandte Chemie, International Edition, 30: 613,1991, and those disclosed by Sanghvi, Y. S., Chapter 15, AntisenseResearch and Applications, pages 289-302, Crooke, S. T. and Lebleu, B.,ed., CRC Press, 1993. Certain of these nucleobases are particularlyuseful for increasing the binding affinity of the compounds of theinvention. These include 5-substituted pyrimidines, 6-azapyrimidines andN-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine,5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutionshave been shown to increase nucleic acid duplex stability by 0.6-1.2° C.and are presently preferred base substitutions, even more particularlywhen combined with 2′-O-methoxyethyl sugar modifications.

Representative United States patents that teach the preparation ofcertain of the above noted modified nucleobases as well as othermodified nucleobases include, but are not limited to, the above notedU.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205; 5,130,302;5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255;5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121,5,596,091; 5,614,617; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and5,681,941, certain of which are commonly owned with the instantapplication, and each of which is herein incorporated by reference, andU.S. Pat. No. 5,750,692, which is commonly owned with the instantapplication and also herein incorporated by reference.

Conjugates

Another modification of the oligonucleotides of the invention involveschemically linking to the oligonucleotide one or more moieties orconjugates which enhance the activity, cellular distribution or cellularuptake of the oligonucleotide. These moieties or conjugates can includeconjugate groups covalently bound to functional groups such as primaryor secondary hydroxyl groups. Conjugate groups of the invention includeintercalators, reporter molecules, polyamines, polyamides, polyethyleneglycols, polyethers, groups that enhance the pharmacodynamic propertiesof oligomers, and groups that enhance the pharmacokinetic properties ofoligomers. Typical conjugate groups include cholesterols, lipids,phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone,acridine, fluoresceins, rhodamines, coumarins, and dyes. Groups thatenhance the pharmacodynamic properties, in the context of thisinvention, include groups that improve uptake, enhance resistance todegradation, and/or strengthen sequence-specific hybridization with thetarget nucleic acid. Groups that enhance the pharmacokinetic properties,in the context of this invention, include groups that improve uptake,distribution, metabolism or excretion of the compounds of the presentinvention. Representative conjugate groups are disclosed inInternational Patent Application PCT/US92/09196, filed Oct. 23, 1992,and U.S. Pat. No. 6,287,860, the entire disclosure of which areincorporated herein by reference. Conjugate moieties include but are notlimited to lipid moieties such as a cholesterol moiety, cholic acid, athioether, e.g., hexyl-5-tritylthiol, a thiocholesterol, an aliphaticchain, e.g., dodecandiol or undecyl residues, a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate, a polyamine or apolyethylene glycol chain, or adamantane acetic acid, a palmityl moiety,or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety.Oligonucleotides of the invention may also be conjugated to active drugsubstances, for example, aspirin, warfarin, phenylbutazone, ibuprofen,suprofen, fenbufen, ketoprofen, (S)-(+)-pranoprofen, carprofen,dansylsarcosine, 2,3,5-triiodobenzoic acid, flufenamic acid, folinicacid, a benzothiadiazide, chlorothiazide, a diazepine, indomethicin, abarbiturate, a cephalosporin, a sulfa drug, an antidiabetic, anantibacterial or an antibiotic. Oligonucleotide-drug conjugates andtheir preparation are described in U.S. patent application Ser. No.09/334,130 (filed Jun. 15, 1999) which is incorporated herein byreference in its entirety. Representative United States patents thatteach the preparation of such oligonucleotide conjugates include, butare not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105;5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941, certain of which are commonly owned with theinstant application, and each of which is herein incorporated byreference.

Chimeric Compounds

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an oligonucleotide.

The present invention also includes antisense compounds which arechimeric compounds. “Chimeric” antisense compounds or “chimeras,” in thecontext of this invention, are antisense compounds, particularlyoligonucleotides, which contain two or more chemically distinct regions,each made up of at least one monomer unit, i.e., a nucleotide in thecase of an oligonucleotide compound. These oligonucleotides typicallycontain at least one region wherein the oligonucleotide is modified soas to confer upon the oligonucleotide increased resistance to nucleasedegradation, increased cellular uptake, increased stability and/orincreased binding affinity for the target nucleic acid. An additionalregion of the oligonucleotide may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNAseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency ofoligonucleotide-mediated inhibition of gene expression. The cleavage ofRNA:RNA hybrids can, in like fashion, be accomplished through theactions of endoribonucleases, such as RNAseL which cleaves both cellularand viral RNA. Cleavage of the RNA target can be routinely detected bygel electrophoresis and, if necessary, associated nucleic acidhybridization techniques known in the art.

Chimeric antisense compounds of the invention may be formed as compositestructures of two or more oligonucleotides, modified oligonucleotides,oligonucleosides and/or oligonucleotide mimetics as described above.Such compounds have also been referred to in the art as hybrids orgapmers. Representative United States patents that teach the preparationof such hybrid structures include, but are not limited to, U.S. Pat.Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711;5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922,certain of which are commonly owned with the instant application, andeach of which is herein incorporated by reference in its entirety.

G. Formulations

The compounds of the invention may also be admixed, encapsulated,conjugated or otherwise associated with other molecules, moleculestructures or mixtures of compounds, as for example, liposomes,receptor-targeted molecules, oral, rectal, topical or otherformulations, for assisting in uptake, distribution and/or absorption.Representative United States patents that teach the preparation of suchuptake, distribution and/or absorption-assisting formulations include,but are not limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;5,580,575; and 5,595,756, each of which is herein incorporated byreference.

The antisense compounds of the invention encompass any pharmaceuticallyacceptable salts, esters, or salts of such esters, or any other compoundwhich, upon administration to an animal, including a human, is capableof providing (directly or indirectly) the biologically active metaboliteor residue thereof. Accordingly, for example, the disclosure is alsodrawn to prodrugs and pharmaceutically acceptable salts of the compoundsof the invention, pharmaceutically acceptable salts of such prodrugs,and other bioequivalents.

The term “prodrug” indicates a therapeutic agent that is prepared in aninactive form that is converted to an active form (i.e., drug) withinthe body or cells thereof by the action of endogenous enzymes or otherchemicals and/or conditions. In particular, prodrug versions of theoligonucleotides of the invention are prepared as SATE[(S-acetyl-2-thioethyl) phosphate] derivatives according to the methodsdisclosed in WO 93/24510 to Gosselin et al., published Dec. 9, 1993 orin WO 94/26764 and U.S. Pat. No. 5,770,713 to Imbach et al.

The term “pharmaceutically acceptable salts” refers to physiologicallyand pharmaceutically acceptable salts of the compounds of the invention:i.e., salts that retain the desired biological activity of the parentcompound and do not impart undesired toxicological effects thereto. Foroligonucleotides, preferred examples of pharmaceutically acceptablesalts and their uses are further described in U.S. Pat. No. 6,287,860,which is incorporated herein in its entirety.

The present invention also includes pharmaceutical compositions andformulations which include the antisense compounds of the invention. Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary, e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Oligonucleotideswith at least one 2′-O-methoxyethyl modification are believed to beparticularly useful for oral administration. Pharmaceutical compositionsand formulations for topical administration may include transdermalpatches, ointments, lotions, creams, gels, drops, suppositories, sprays,liquids and powders. Conventional pharmaceutical carriers, aqueous,powder or oily bases, thickeners and the like may be necessary ordesirable. Coated condoms, gloves and the like may also be useful.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, foams and liposome-containingformulations. The pharmaceutical compositions and formulations of thepresent invention may comprise one or more penetration enhancers,carriers, excipients or other active or inactive ingredients.

Emulsions are typically heterogenous systems of one liquid dispersed inanother in the form of droplets usually exceeding 0.1 μm in diameter.Emulsions may contain additional components in addition to the dispersedphases, and the active drug which may be present as a solution in eitherthe aqueous phase, oily phase or itself as a separate phase.Microemulsions are included as an embodiment of the present invention.Emulsions and their uses are well known in the art and are furtherdescribed in U.S. Pat. No. 6,287,860, which is incorporated herein inits entirety.

Formulations of the present invention include liposomal formulations. Asused in the present invention, the term “liposome” means a vesiclecomposed of amphiphilic lipids arranged in a spherical bilayer orbilayers. Liposomes are unilamellar or multilamellar vesicles which havea membrane formed from a lipophilic material and an aqueous interiorthat contains the composition to be delivered. Cationic liposomes arepositively charged liposomes which are believed to interact withnegatively charged DNA molecules to form a stable complex. Liposomesthat are pH-sensitive or negatively-charged are believed to entrap DNArather than complex with it. Both cationic and noncationic liposomeshave been used to deliver DNA to cells.

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome comprises oneor more glycolipids or is derivatized with one or more hydrophilicpolymers, such as a polyethylene glycol (PEG) moiety. Liposomes andtheir uses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

The pharmaceutical formulations and compositions of the presentinvention may also include surfactants. The use of surfactants in drugproducts, formulations and in emulsions is well known in the art.Surfactants and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety.

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly oligonucleotides. In addition to aiding the diffusion ofnon-lipophilic drugs across cell membranes, penetration enhancers alsoenhance the permeability of lipophilic drugs. Penetration enhancers maybe classified as belonging to one of five broad categories, i.e.,surfactants, fatty acids, bile salts, chelating agents, andnon-chelating non-surfactants. Penetration enhancers and their uses arefurther described in U.S. Pat. No. 6,287,860, which is incorporatedherein in its entirety.

One of skill in the art will recognize that formulations are routinelydesigned according to their intended use, i.e. route of administration.

Preferred formulations for topical administration include those in whichthe oligonucleotides of the invention are in admixture with a topicaldelivery agent such as lipids, liposomes, fatty acids, fatty acidesters, steroids, chelating agents and surfactants. Preferred lipids andliposomes include neutral (e.g. dioleoylphosphatidyl DOPE ethanolamine,dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline)negative (e.g. dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g.dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidylethanolamine DOTMA).

For topical or other administration, oligonucleotides of the inventionmay be encapsulated within liposomes or may form complexes thereto, inparticular to cationic liposomes. Alternatively, oligonucleotides may becomplexed to lipids, in particular to cationic lipids. Preferred fattyacids and esters, pharmaceutically acceptable salts thereof, and theiruses are further described in U.S. Pat. No. 6,287,860, which isincorporated herein in its entirety.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. Preferred oral formulationsare those in which oligonucleotides of the invention are administered inconjunction with one or more penetration enhancers surfactants andchelators. Preferred surfactants include fatty acids and/or esters orsalts thereof, bile acids and/or salts thereof. Preferred bileacids/salts and fatty acids and their uses are further described in U.S.Pat. No. 6,287,860, which is incorporated herein in its entirety. Alsopreferred are combinations of penetration enhancers, for example, fattyacids/salts in combination with bile acids/salts. A particularlypreferred combination is the sodium salt of lauric acid, capric acid andUDCA. Further penetration enhancers include polyoxyethylene-9-laurylether, polyoxyethylene-20-cetyl ether. Oligonucleotides of the inventionmay be delivered orally, in granular form including sprayed driedparticles, or complexed to form micro or nanoparticles. Oligonucleotidecomplexing agents and their uses are further described in U.S. Pat. No.6,287,860, which is incorporated herein in its entirety. Oralformulations for oligonucleotides and their preparation are described indetail in U.S. application Ser. Nos. 09/108,673 (filed Jul. 1, 1998),09/315,298 (filed May 20, 1999) and 10/071,822, filed Feb. 8, 2002, eachof which is incorporated herein by reference in their entirety.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionswhich may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Certain embodiments of the invention provide pharmaceutical compositionscontaining one or more oligomeric compounds and one or more otherchemotherapeutic agents which function by a non-antisense mechanism.Examples of such chemotherapeutic agents include but are not limited tocancer chemotherapeutic drugs such as daunorubicin, daunomycin,dactinomycin, doxorubicin, epirubicin, idarubicin, esorubicin,bleomycin, mafosfamide, ifosfamide, cytosine arabinoside,bis-chloroethylnitrosurea, busulfan, mitomycin C, actinomycin D,mithramycin, prednisone, hydroxyprogesterone, testosterone, tamoxifen,dacarbazine, procarbazine, hexamethylmelamine, pentamethylmelamine,mitoxantrone, amsacrine, chlorambucil, methylcyclohexylnitrosurea,nitrogen mustards, melphalan, cyclophosphamide, 6-mercaptopurine,6-thioguanine, cytarabine, 5-azacytidine, hydroxyurea, deoxycoformycin,4-hydroxyperoxycyclophosphoramide, 5-fluorouracil (5-FU),5-fluorodeoxyuridine (5-FUdR), methotrexate (MTX), colchicine, taxol,vincristine, vinblastine, etoposide (VP-16), trimetrexate, irinotecan,topotecan, gemcitabine, teniposide, cisplatin and diethylstilbestrol(DES). When used with the compounds of the invention, suchchemotherapeutic agents may be used individually (e.g., 5-FU andoligonucleotide), sequentially (e.g., 5-FU and oligonucleotide for aperiod of time followed by MTX and oligonucleotide), or in combinationwith one or more other such chemotherapeutic agents (e.g., 5-FU, MTX andoligonucleotide, or 5-FU, radiotherapy and oligonucleotide).Anti-inflammatory drugs, including but not limited to nonsteroidalanti-inflammatory drugs and corticosteroids, and antiviral drugs,including but not limited to ribivirin, vidarabine, acyclovir andganciclovir, may also be combined in compositions of the invention.Combinations of antisense compounds and other non-antisense drugs arealso within the scope of this invention. Two or more combined compoundsmay be used together or sequentially.

In another related embodiment, compositions of the invention may containone or more antisense compounds, particularly oligonucleotides, targetedto a first nucleic acid and one or more additional antisense compoundstargeted to a second nucleic acid target. Alternatively, compositions ofthe invention may contain two or more antisense compounds targeted todifferent regions of the same nucleic acid target. Numerous examples ofantisense compounds are known in the art. Two or more combined compoundsmay be used together or sequentially.

H. Dosing

The formulation of therapeutic compositions and their subsequentadministration (dosing) is believed to be within the skill of those inthe art. Dosing is dependent on severity and responsiveness of thedisease state to be treated, with the course of treatment lasting fromseveral days to several months, or until a cure is effected or adiminution of the disease state is achieved. Optimal dosing schedulescan be calculated from measurements of drug accumulation in the body ofthe patient. Persons of ordinary skill can easily determine optimumdosages, dosing methodologies and repetition rates. Optimum dosages mayvary depending on the relative potency of individual oligonucleotides,and can generally be estimated based on EC₅₀s found to be effective inin vitro and in vivo animal models. In general, dosage is from 0.01 ugto 100 g per kg of body weight, and may be given once or more daily,weekly, monthly or yearly, or even once every 2 to 20 years. Persons ofordinary skill in the art can easily estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the patient undergo maintenance therapy to prevent therecurrence of the disease state, wherein the oligonucleotide isadministered in maintenance doses, ranging from 0.01 ug to 100 g per kgof body weight, once or more daily, to once every 20 years.

While the present invention has been described with specificity inaccordance with certain of its preferred embodiments, the followingexamples serve only to illustrate the invention and are not intended tolimit the same.

EXAMPLES Example 1 Synthesis of Nucleoside Phosphoramidites

The following compounds, including amidites and their intermediates wereprepared as described in U.S. Pat. No. 6,426,220 and published PCT WO02/36743; 5′-O -Dimethoxytrityl-thymidine intermediate for 5-methyl dCamidite, 5′-O-Dimethoxytrityl-2′-deoxy-5-methylcytidine intermediate for5-methyl-dC amidite, 5′-O-Dimethoxytrityl-2′-deoxy-N4-benzoyl-5-methylcytidine penultimateintermediate for 5-methyl dC amidite,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-deoxy-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(5-methyl dC amidite), 2′-Fluorodeoxyadenosine, 2′-Fluorodeoxyguanosine,2′-Fluorouridine, 2′-Fluorodeoxycytidine, 2′-O-(2-Methoxyethyl) modifiedamidites, 2′-O-(2-methoxyethyl)-5-methyluridine intermediate,5′-O-DMT-2′-O-(2-methoxyethyl)-5-methyluridine penultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-5-methyluridin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE T amidite),5′-O-Dimethoxytrityl-2′-O-(2-methoxyethyl)-5-methylcytidineintermediate,5′-O-dimethoxytrityl-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methyl-cytidinepenultimate intermediate,[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-benzoyl-5-methylcytidin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE 5-Me-C amidite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁶-benzoyladenosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE A amdite),[5′-O-(4,4′-Dimethoxytriphenylmethyl)-2′-O-(2-methoxyethyl)-N⁴-isobutyrylguanosin-3′-O-yl]-2-cyanoethyl-N,N-diisopropylphosphoramidite(MOE G amidite), 2′-O-(Aminooxyethyl) nucleoside amidites and 2′-O-(dimethylaminooxyethyl) nucleoside amidites,2′-(Dimethylaminooxyethoxy) nucleoside amidites, 5′-O-tert-Butyldiphenylsilyl-O²-2′-anhydro-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-(2-hydroxyethyl)-5-methyluridine,2′-O-([2-phthalimidoxy)ethyl]-5′-t-butyldiphenylsilyl-5-methyluridine,5′-O-tert-butyldiphenylsilyl-2′-O-[(2-formadoximinooxy)ethyl]-5-methyluridine,5′-O-tert-Butyldiphenylsilyl-2′-O-[N,Ndimethylaminooxyethyl]-5-methyluridine,2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(dimethylaminooxyethyl)-5-methyluridine,5′-O-DMT-2′-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-(Aminooxyethoxy) nucleoside amidites,N2-isobutyryl-6-O-diphenylcarbamoyl-2′-O-(2-ethylacetyl)-5′-O-(4,4′-dimethoxytrityl)guanosine-3′-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite],2′-dimethylaminoethoxyethoxy (2′-DMAEOE) nucleoside amidites,2′-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl uridine,5′-O-dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl uridine and 5′-O-Dimethoxytrityl-2′-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyluridine-3′-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite.

Example 2 Oligonucleotide and Oligonucleoside Synthesis

The antisense compounds used in accordance with this invention may beconveniently and routinely made through the well-known technique ofsolid phase synthesis. Equipment for such synthesis is sold by severalvendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is well known to usesimilar techniques to prepare oligonucleotides such as thephosphorothioates and alkylated derivatives.

Oligonucleotides: Unsubstituted and substituted phosphodiester (P═O)oligonucleotides are synthesized on an automated DNA synthesizer(Applied Biosystems model 394) using standard phosphoramidite chemistrywith oxidation by iodine.

Phosphorothioates (P═S) are synthesized similar to phosphodiesteroligonucleotides with the following exceptions: thiation was effected byutilizing a 10% w/v solution of 3,H-1,2-benzodithiole-3-one 1,1-dioxidein acetonitrile for the oxidation of the phosphite linkages. Thethiation reaction step time was increased to 180 sec and preceded by thenormal capping step. After cleavage from the CPG column and deblockingin concentrated ammonium hydroxide at 55° C. (12-16 hr), theoligonucleotides were recovered by precipitating with >3 volumes ofethanol from a 1 M NH₄OAc solution. Phosphinate oligonucleotides areprepared as described in U.S. Pat. No. 5,508,270, herein incorporated byreference.

Alkyl phosphonate oligonucleotides are prepared as described in U.S.Pat. No. 4,469,863, herein incorporated by reference.

3′-Deoxy-3′-methylene phosphonate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,610,289 or 5,625,050, herein incorporatedby reference.

Phosphoramidite oligonucleotides are prepared as described in U.S. Pat.Nos. 5,256,775 or 5,366,878, herein incorporated by reference.

Alkylphosphonothioate oligonucleotides are prepared as described inpublished PCT applications PCT/US94/00902 and PCT/US93/06976 (publishedas WO 94/17093 and WO 94/02499, respectively), herein incorporated byreference.

3′-Deoxy-3′-amino phosphoramidate oligonucleotides are prepared asdescribed in U.S. Pat. No. 5,476,925, herein incorporated by reference.

Phosphotriester oligonucleotides are prepared as described in U.S. Pat.No. 5,023,243, herein incorporated by reference.

Borano phosphate oligonucleotides are prepared as described in U.S. Pat.Nos. 5,130,302 and 5,177,198, both herein incorporated by reference.

Oligonucleosides: Methylenemethylimino linked oligonucleosides, alsoidentified as MMI linked oligonucleosides, methylenedimethylhydrazolinked oligonucleosides, also identified as MDH linked oligonucleosides,and methylenecarbonylamino linked oligonucleosides, also identified asamide-3 linked oligonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified as amide-4 linked oligo-nucleosides,as well as mixed backbone compounds having, for instance, alternatingMMI and P═O or P═S linkages are prepared as described in U.S. Pat. Nos.5,378,825, 5,386,023, 5,489,677, 5,602,240 and 5,610,289, all of whichare herein incorporated by reference.

Formacetal and thioformacetal linked oligonucleosides are prepared asdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein incorporatedby reference.

Ethylene oxide linked oligonucleosides are prepared as described in U.S.Pat. No. 5,223,618, herein incorporated by reference.

Example 3 RNA Synthesis

In general, RNA synthesis chemistry is based on the selectiveincorporation of various protecting groups at strategic intermediaryreactions. Although one of ordinary skill in the art will understand theuse of protecting groups in organic synthesis, a useful class ofprotecting groups includes silyl ethers. In particular bulky silylethers are used to protect the 5′-hydroxyl in combination with anacid-labile orthoester protecting group on the 2′-hydroxyl. This set ofprotecting groups is then used with standard solid-phase synthesistechnology. It is important to lastly remove the acid labile orthoesterprotecting group after all other synthetic steps. Moreover, the earlyuse of the silyl protecting groups during synthesis ensures facileremoval when desired, without undesired deprotection of 2′ hydroxyl.

Following this procedure for the sequential protection of the5′-hydroxyl in combination with protection of the 2′-hydroxyl byprotecting groups that are differentially removed and are differentiallychemically labile, RNA oligonucleotides were synthesized.

RNA oligonucleotides are synthesized in a stepwise fashion. Eachnucleotide is added sequentially (3′- to 5′-direction) to a solidsupport-bound oligonucleotide. The first nucleoside at the 3′-end of thechain is covalently attached to a solid support. The nucleotideprecursor, a ribonucleoside phosphoramidite, and activator are added,coupling the second base onto the 5′-end of the first nucleoside. Thesupport is washed and any unreacted 5′-hydroxyl groups are capped withacetic anhydride to yield 5′-acetyl moieties. The linkage is thenoxidized to the more stable and ultimately desired P(V) linkage. At theend of the nucleotide addition cycle, the 5′-silyl group is cleaved withfluoride. The cycle is repeated for each subsequent nucleotide.

Following synthesis, the methyl protecting groups on the phosphates arecleaved in 30 minutes utilizing 1 Mdisodium-2-carbamoyl-2-cyanoethylene-1,1-dithiolate trihydrate (S₂Na₂)in DMF. The deprotection solution is washed from the solid support-boundoligonucleotide using water. The support is then treated with 40%methylamine in water for 10 minutes at 55° C. This releases the RNAoligonucleotides into solution, deprotects the exocyclic amines, andmodifies the 2′-groups. The oligonucleotides can be analyzed by anionexchange HPLC at this stage.

The 2′-orthoester groups are the last protecting groups to be removed.The ethylene glycol monoacetate orthoester protecting group developed byDharmacon Research, Inc. (Lafayette, Colo.), is one example of a usefulorthoester protecting group which, has the following importantproperties. It is stable to the conditions of nucleoside phosphoramiditesynthesis and oligonucleotide synthesis. However, after oligonucleotidesynthesis the oligonucleotide is treated with methylamine which not onlycleaves the oligonucleotide from the solid support but also removes theacetyl groups from the orthoesters. The resulting 2-ethyl-hydroxylsubstituents on the orthoester are less electron withdrawing than theacetylated precursor. As a result, the modified orthoester becomes morelabile to acid-catalyzed hydrolysis. Specifically, the rate of cleavageis approximately 10 times faster after the acetyl groups are removed.Therefore, this orthoester possesses sufficient stability in order to becompatible with oligonucleotide synthesis and yet, when subsequentlymodified, permits deprotection to be carried out under relatively mildaqueous conditions compatible with the final RNA oligonucleotideproduct.

Additionally, methods of RNA synthesis are well known in the art(Scaringe, Ph.D. Thesis, University of Colorado, 1996; Scaringe et al.,J. Am. Chem. Soc. 120: 11820-11821; 1998; Matteucci and Caruthers, J.Am. Chem. Soc. 103: 3185-3191, 1981; Beaucage and Caruthers, TetrahedronLett. 22: 1859-1862, 1981; Dahl et al., Acta Chem. Scand. 44: 639-641;1990, Reddy et al., Tetrahedrom Lett. 25: 4311-4314, 1994; Wincott etal., Nucleic Acids Res. 23: 2677-2684, 1995; Griffin et al., Tetrahedron23: 2301-2313, 1967a; Griffin et al., Tetrahedron 23: 2315-2331, 1967b).

RNA antisense compounds (RNA oligonucleotides) of the present inventioncan be synthesized by the methods herein or purchased from DharmaconResearch, Inc (Lafayette, Colo.). Once synthesized, complementary RNAantisense compounds can then be annealed by methods known in the art toform double stranded (duplexed) antisense compounds. For example,duplexes can be formed by combining 30 μl of each of the complementarystrands of RNA oligonucleotides (50 uM RNA oligonucleotide solution) and15 μl of 5× annealing buffer (100 mM potassium acetate, 30 mM HEPES-KOHpH 7.4, 2 mM magnesium acetate) followed by heating for 1 minute at 90°C., then 1 hour at 37° C. The resulting duplexed antisense compounds canbe used in kits, assays, screens, or other methods to investigate therole of a target nucleic acid.

Example 4 Synthesis of Chimeric Oligonucleotides

Chimeric oligonucleotides, oligonucleosides or mixedoligonucleotides/oligonucleosides of the invention can be of severaldifferent types. These include a first type wherein the “gap” segment oflinked nucleosides is positioned between 5′ and 3′ “wing” segments oflinked nucleosides and a second “open end” type wherein the “gap”segment is located at either the 3′ or the 5′ terminus of the oligomericcompound. Oligonucleotides of the first type are also known in the artas “gapmers” or gapped oligonucleotides. Oligonucleotides of the secondtype are also known in the art as “hemimers” or “wingmers”.

[2′-O-Me]-[2′-deoxy]-[2′-O-Me] Chimeric PhosphorothioateOligonucleotides

Chimeric oligonucleotides having 2′-O-alkyl phosphorothioate and2′-deoxy phosphorothioate oligonucleotide segments are synthesized usingan Applied Biosystems automated DNA synthesizer Model 394, as above.Oligonucleotides are synthesized using the automated synthesizer and2′-deoxy-5′-dimethoxytrityl-3′-O-phosphoramidite for the DNA portion and5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite for 5′ and 3′ wings.The standard synthesis cycle is modified by incorporating coupling stepswith increased reaction times for the5′-dimethoxytrityl-2′-O-methyl-3′-O-phosphoramidite. The fully protectedoligonucleotide is cleaved from the support and deprotected inconcentrated ammonia (NH₄OH) for 12-16 hr at 55° C. The deprotectedoligo is then recovered by an appropriate method (precipitation, columnchromatography, volume reduced in vacuo and analyzedspetrophotometrically for yield and for purity by capillaryelectrophoresis and by mass spectrometry.

[2′-O-(2-Methoxyethyl)]-[2′-deoxy]-[2′-O-(Methoxyethyl)] ChimericPhosphorothioate Oligonucleotides

[2′-O-(2-methoxyethyl)]-[2′-deoxy]-[-2′-O-(methoxyethyl)] chimericphosphorothioate oligonucleotides were prepared as per the procedureabove for the 2′-O-methyl chimeric oligonucleotide, with thesubstitution of 2′-O-(methoxyethyl) amidites for the 2′-O-methylamidites.

[2′-O-(2-Methoxyethyl)Phosphodiester]-[2′-deoxyPhosphorothioate]-[2′-O-(2-Methoxyethyl) Phosphodiester] ChimericOligonucleotides

[2′-O-(2-methoxyethyl phosphodiester]-[2′-deoxyphosphorothioate]-[2′-0-(methoxyethyl) phosphodiester] chimericoligonucleotides are prepared as per the above procedure for the2′-O-methyl chimeric oligonucleotide with the substitution of2′-O-(methoxyethyl) amidites for the 2′-O-methyl amidites, oxidationwith iodine to generate the phosphodiester internucleotide linkageswithin the wing portions of the chimeric structures and sulfurizationutilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) togenerate the phosphorothioate internucleotide linkages for the centergap.

Other chimeric oligonucleotides, chimeric oligonucleosides and mixedchimeric oligonucleotides/oligonucleosides are synthesized according toU.S. Pat. No. 5,623,065, herein incorporated by reference.

Example 5 Design and Screening of Duplexed Antisense Compounds TargetingIGF-1R mRNA

In accordance with the present invention, a series of nucleic acidduplexes comprising the antisense compounds of the present invention andtheir complements can be designed to target IGF-IR mRNA. The nucleobasesequence of the antisense strand of the duplex comprises at least aportion of an oligonucleotide selected from SEQ ID NOs: 1 through 76 andSEQ ID NO: 100 through 136 shown in Table 1 including preferred ASO'sISIS 175308, 175302, 175314, 175307, 175317, 175323, 232744, 323747,323767, 323762 and 323737. The ends of the strands may be modified bythe addition of one or more natural or modified nucleobases to form anoverhang. The sense strand of the dsRNA is then designed and synthesizedas the complement of the antisense strand and may also containmodifications or additions to either terminus. For example, in oneembodiment, both strands of the dsRNA duplex would be complementary overthe central nucleobases, each having overhangs at one or both termini.

For example, a duplex comprising an antisense strand having the sequenceCGAGAGGCGGACGGGACCG and having a two-nucleobase overhang ofdeoxythymidine(dT) would have the following structure:

RNA strands of the duplex can be synthesized by methods disclosed hereinor purchased from Dharmacon Research Inc., (Lafayette, Colo.). Oncesynthesized, the complementary strands are annealed. The single strandsare aliquoted and diluted to a concentration of 50 uM. Once diluted, 30uL of each strand is combined with 15 uL of a 5× solution of annealingbuffer. The final concentration of said buffer is 100 mM potassiumacetate, 30 mM HEPES-KOH pH 7.4, and 2 mM magnesium acetate. The finalvolume is 75 uL. This solution is incubated for 1 minute at 90° C. andthen centrifuged for 15 seconds. The tube is allowed to sit for 1 hourat 37° C. at which time the dsRNA duplexes are used in experimentation.The final concentration of the dsRNA duplex is 20 uM. This solution canbe stored frozen (−20° C.) and freeze-thawed up to 5 times.

Once prepared, the duplexed antisense compounds are evaluated for theirability to modulate IGF-IR gene expression.

When cells reached 80% confluency, they are treated with duplexedantisense compounds of the invention. For cells grown in 96-well plates,wells are washed once with 200 μL OPTI-MEM-1 reduced-serum medium (GibcoBRL) and then treated with 130 μL of OPTI-MEM-1 containing 12 μg/mLLIPOFECTIN (Gibco BRL) and the desired duplex antisense compound at afinal concentration of 200 nM. After 5 hours of treatment, the medium isreplaced with fresh medium. Cells are harvested 16 hours aftertreatment, at which time RNA is isolated and target reduction measuredby RT-PCR.

Example 6 Oligonucleotide Isolation

After cleavage from the controlled pore glass solid support anddeblocking in concentrated ammonium hydroxide at 55° C. for 12-16 hours,the oligonucleotides or oligonucleosides are recovered by precipitationout of 1 M NH₄OAc with >3 volumes of ethanol. Synthesizedoligonucleotides were analyzed by electrospray mass spectroscopy(molecular weight determination) and by capillary gel electrophoresisand judged to be at least 70% full length material. The relative amountsof phosphorothioate and phosphodiester linkages obtained in thesynthesis was determined by the ratio of correct molecular weightrelative to the −16 amu product (+/−32 +/−48). For some studiesoligonucleotides were purified by HPLC, as described by Chiang et al.,J. Biol. Chem. 266: 18162-18171, 1991. Results obtained withHPLC-purified material were similar to those obtained with non-HPLCpurified material.

Example 7 Oligonucleotide Synthesis—96 Well Plate Format

Oligonucleotides were synthesized via solid phase P(III) phosphoramiditechemistry on an automated synthesizer capable of assembling 96 sequencessimultaneously in a 96-well format. Phosphodiester internucleotidelinkages were afforded by oxidation with aqueous iodine.Phosphorothioate internucleotide linkages were generated bysulfurization utilizing 3,H-1,2 benzodithiole-3-one 1,1 dioxide(Beaucage Reagent) in anhydrous acetonitrile. Standard base-protectedbeta-cyanoethyl-diiso-propyl phosphoramidites were purchased fromcommercial vendors (e.g. PE-Applied Biosystems, Foster City, Calif., orPharmacia, Piscataway, N.J.). Non-standard nucleosides are synthesizedas per standard or patented methods. They are utilized as base protectedbeta-cyanoethyldiisopropyl phosphoramidites.

Oligonucleotides were cleaved from support and deprotected withconcentrated NH₄OH at elevated temperature (55-60° C.) for 12-16 hoursand the released product then dried in vacuo. The dried product was thenre-suspended in sterile water to afford a master plate from which allanalytical and test plate samples are then diluted utilizing roboticpipettors.

Example 8 Oligonucleotide Analysis—96-Well Plate Format

The concentration of oligonucleotide in each well was assessed bydilution of samples and UV absorption spectroscopy. The full-lengthintegrity of the individual products was evaluated by capillaryelectrophoresis (CE) in either the 96-well format (Beckman P/ACE(trademark) MDQ) or, for individually prepared samples, on a commercialCE apparatus (e.g., Beckman P/ACE (trademark) 5000, ABI 270). Base andbackbone composition was confirmed by mass analysis of the compoundsutilizing electrospray-mass spectroscopy. All assay test plates werediluted from the master plate using single and multi-channel roboticpipettors. Plates were judged to be acceptable if at least 85% of thecompounds on the plate were at least 85% full length.

Example 9 Cell Culture and Oligonucleotide Treatment

The effect of antisense compounds on target nucleic acid expression canbe tested in any of a variety of cell types provided that the targetnucleic acid is present at measurable levels. This can be routinelydetermined using, for example, PCR or Northern blot analysis. Thefollowing cell types are provided for illustrative purposes, but othercell types can be routinely used, provided that the target is expressedin the cell type chosen. This can be readily determined by methodsroutine in the art, for example Northern blot analysis, ribonucleaseprotection assays, or RT-PCR.

T-24 Cells:

The human transitional cell bladder carcinoma cell line T-24 wasobtained from the American Type Culture Collection (ATCC) (Manassas,Va.). T-24 cells were routinely cultured in complete McCoy's 5A basalmedia (Invitrogen Corporation, Carlsbad, Calif.) supplemented with 10%w/v fetal calf serum (Invitrogen Corporation, Carlsbad, Calif.),penicillin 100 units per mL, and streptomycin 100 micrograms per mL(Invitrogen Corporation, Carlsbad, Calif.). Cells were routinelypassaged by trypsinization and dilution when they reached 90%confluence. Cells were seeded into 96-well plates (Falcon-Primaria#353872) at a density of 7000 cells/well for use in RT-PCR analysis. ForNorthern blotting or other analysis, cells may be seeded onto 100 mm orother standard tissue culture plates and treated similarly, usingappropriate volumes of medium and oligonucleotide.

A549 Cells:

The human lung carcinoma cell line A549 was obtained from the AmericanType Culture Collection (ATCC) (Manassas, Va.). A549 cells wereroutinely cultured in DMEM basal media (Invitrogen Corporation,Carlsbad, Calif.) supplemented with 10% fetal calf serum (InvitrogenCorporation, Carlsbad, Calif.), penicillin 100 units per mL, andstreptomycin 100 micrograms per mL (Invitrogen Corporation, Carlsbad,Calif.). Cells were routinely passaged by trypsinization and dilutionwhen they reached 90% confluence.

NHDF Cells:

Human neonatal dermal fibroblast (NHDF) were obtained from the CloneticsCorporation (Walkersville, Md.). NHDFs were routinely maintained inFibroblast Growth Medium (Clonetics Corporation, Walkersville, Md.)supplemented as recommended by the supplier. Cells were maintained forup to 10 passages as recommended by the supplier.

HEK Cells:

Human embryonic keratinocytes (HEK) were obtained from the CloneticsCorporation (Walkersville, Md.). HEKs were routinely maintained inKeratinocyte Growth Medium (Clonetics Corporation, Walkersville, Md.)formulated as recommended by the supplier. Cells were routinelymaintained for up to 10 passages as recommended by the supplier.

Treatment with Antisense Compounds

When cells reached 65-75% confluency, they were treated witholigonucleotide. For cells grown in 96-well plates, wells were washedonce with 100 μL OPTI-MEM (trademark)-l reduced-serum medium (InvitrogenCorporation, Carlsbad, Calif.) and then treated with 130 μL of OPTI-MEM(trademark)-1 containing 3.75 μg/mL LIPOFECTIN (trademark) (InvitrogenCorporation, Carlsbad, Calif.) and the desired concentration ofoligonucleotide. Cells are treated and data are obtained in triplicate.After 4-7 hours of treatment at 37° C., the medium was replaced withfresh medium. Cells were harvested 16-24 hours after oligonucleotidetreatment.

The concentration of oligonucleotide used varies from cell line to cellline. To determine the optimal oligonucleotide concentration for aparticular cell line, the cells are treated with a positive controloligonucleotide at a range of concentrations. For human cells thepositive control oligonucleotide is selected from either ISIS 13920(TCCGTCATCGCTCCTCAGGG, SEQ ID NO:79) which is targeted to human H-ras,or ISIS 18078, (GTGCGCGCGAGCCCGAAATC, SEQ ID NO:80) which is targeted tohuman Jun-N-terminal kinase-2 (JNK2). Both controls are2′-O-methoxyethyl gapmers (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone. For mouse or rat cells the positive controloligonucleotide is ISIS 15770, ATGCATTCTGCCCCCAAGGA, SEQ ID NO:81, a2′-O-methoxyethyl gapmer (2′-O-methoxyethyls shown in bold) with aphosphorothioate backbone which is targeted to both mouse and rat c-raf.The concentration of positive control oligonucleotide that results in80% inhibition of c-H-ras (for ISIS 13920), JNK2 (for ISIS 18078) orc-raf (for ISIS 15770) mRNA is then utilized as the screeningconcentration for new oligonucleotides in subsequent experiments forthat cell line. If 80% inhibition is not achieved, the lowestconcentration of positive control oligonucleotide that results in 60%inhibition of c-H-ras, JNK2 or c-raf mRNA is then utilized as theoligonucleotide screening concentration in subsequent experiments forthat cell line. If 60% inhibition is not achieved, that particular cellline is deemed as unsuitable for oligonucleotide transfectionexperiments. The concentrations of antisense oligonucleotides usedherein are from 50 nM to 300 nM.

Example 10 Analysis of Oligonucleotide Inhibition of IGF-1R GeneExpression

Antisense modulation of IGF-1R gene expression can be assayed in avariety of ways known in the art. For example, IGF-IR mRNA levels can bequantitated by, e.g., Northern blot analysis, competitive polymerasechain reaction (PCR), or real-time PCR (RT-PCR). Real-time quantitativePCR is presently preferred. RNA analysis can be performed on totalcellular RNA or poly(A)+ mRNA. The preferred method of RNA analysis ofthe present invention is the use of total cellular RNA as described inother examples herein. Methods of RNA isolation are well known in theart. Northern blot analysis is also routine in the art. Real-timequantitative (PCR) can be conveniently accomplished using thecommercially available ABI PRISM (trademark) 7600, 7700, or 7900Sequence Detection System, available from PE-Applied Biosystems, FosterCity, Calif. and used according to manufacturer's instructions.

Protein levels of IGF-IR can be quantitated in a variety of ways wellknown in the art, such as immunoprecipitation, Western blot analysis(immunoblotting), enzyme-linked immunosorbent assay (ELISA) orfluorescence-activated cell sorting (FACS). Antibodies directed toIGF-IR can be identified and obtained from a variety of sources, such asthe MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.),or can be prepared via conventional monoclonal or polyclonal antibodygeneration methods well known in the art.

Example 11 Design of Phenotypic Assays and In Vivo Studies for the Useof IGF-1R Gene Expression Inhibitors

Phenotypic Assays

Once IGF-IR gene expression inhibitors have been identified by themethods disclosed herein, the compounds are further investigated in oneor more phenotypic assays, each having measurable endpoints predictiveof efficacy in the treatment of a particular disease state or condition.

Phenotypic assays, kits and reagents for their use are well known tothose skilled in the art and are herein used to investigate the roleand/or association of IGF-IR in health and disease. Representativephenotypic assays, which can be purchased from any one of severalcommercial vendors, include those for determining cell viability,cytotoxicity, proliferation or cell survival (Molecular Probes, Eugene,Oreg.; PerkinElmer, Boston, Mass.), protein-based assays includingenzymatic assays (Panvera, LLC, Madison, Wis.; BD Biosciences, FranklinLakes, N.J.; Oncogene Research Products, San Diego, Calif.), cellregulation, signal transduction, inflammation, oxidative processes andapoptosis (Assay Designs Inc., Ann Arbor, Mich.), triglycerideaccumulation (Sigma-Aldrich, St. Louis, Mo.), angiogenesis assays, tubeformation assays, cytokine and hormone assays and metabolic assays(Chemicon International Inc., Temecula, Calif.; Amersham Biosciences,Piscataway, N.J.).

In one non-limiting example, cells determined to be appropriate for aparticular phenotypic assay (i.e., MCF-7 cells selected for breastcancer studies; adipocytes for obesity studies) are treated with IGF-IRgene expression inhibitors identified from the in vitro studies as wellas control compounds at optimal concentrations which are determined bythe methods described above. At the end of the treatment period, treatedand untreated cells are analyzed by one or more methods specific for theassay to determine phenotypic outcomes and endpoints.

Phenotypic endpoints include changes in cell morphology over time ortreatment dose as well as changes in levels of cellular components suchas proteins, lipids, nucleic acids, hormones, saccharides or metals.Measurements of cellular status which include pH, stage of the cellcycle, intake or excretion of biological indicators by the cell, arealso endpoints of interest.

Analysis of the geneotype of the cell (measurement of the expression ofone or more of the genes of the cell) after treatment is also used as anindicator of the efficacy or potency of the IGF-IR gene expressioninhibitors. Hallmark genes, or those genes suspected to be associatedwith a specific disease state, condition, or phenotype, are measured inboth treated and untreated cells.

In Vivo Studies

The individual subjects of the in vivo studies described herein arewarm-blooded vertebrate animals, which includes humans.

The clinical trial is subjected to rigorous controls to ensure thatindividuals are not unnecessarily put at risk and that they are fullyinformed about their role in the study. To account for the psychologicaleffects of receiving treatments, volunteers are randomly given placeboor IGF-IR gene expression inhibitor. Furthermore, to prevent the doctorsfrom being biased in treatments, they are not informed as to whether themedication they are administering is a IGF-IR gene expression inhibitoror a placebo. Using this randomization approach, each volunteer has thesame chance of being given either the new treatment or the placebo.

Volunteers receive either the IGF-IR gene expression inhibitor orplacebo for eight week period with biological parameters associated withthe indicated disease state or condition being measured at the beginning(baseline measurements before any treatment), end (after the finaltreatment), and at regular intervals during the study period. Suchmeasurements include the levels of nucleic acid molecules encodingIGF-IR or IGF-IR protein levels in body fluids, tissues or organscompared to pre-treatment levels. Other measurements include, but arenot limited to, indices of the disease state or condition being treated,body weight, blood pressure, serum titers of pharmacologic indicators ofdisease or toxicity as well as ADME (absorption, distribution,metabolism and excretion) measurements.

Information recorded for each patient includes age (years), gender,height (cm), family history of disease state or condition (yes/no),motivation rating (some/moderate/great) and number and type of previoustreatment regimens for the indicated disease or condition.

Volunteers taking part in this study are healthy adults (age 18 to 65years) and roughly an equal number of males and females participate inthe study. Volunteers with certain characteristics are equallydistributed for placebo and IGF-IR gene expression inhibitor treatment.In general, the volunteers treated with placebo have little or noresponse to treatment, whereas the volunteers treated with the IGF-IRgene expression inhibitor show positive trends in their disease state orcondition index at the conclusion of the study.

Example 12 RNA Isolation

Poly(A)+ mRNA Isolation

Poly(A)+ mRNA was isolated according to Miura et al. (Clin. Chem. 42:1758-1764, 1996). Other methods for poly(A)+ mRNA isolation are routinein the art. Briefly, for cells grown on 96-well plates, growth mediumwas removed from the cells and each well was washed with 200 μL coldPBS. 60 μL lysis buffer (10 mM Tris-HCl, pH 7.6, 1 mM EDTA, 0.5 M NaCl,0.5% NP-40, 20 mM vanadyl-ribonucleoside complex) was added to eachwell, the plate was gently agitated and then incubated at roomtemperature for five minutes. 55 μL of lysate was transferred to Oligod(T) coated 96-well plates (AGCT Inc., Irvine Calif.). Plates wereincubated for 60 minutes at room temperature, washed 3 times with 200 μLof wash buffer (10 mM Tris-HCl pH 7.6, 1 mM EDTA, 0.3 M NaCl). After thefinal wash, the plate was blotted on paper towels to remove excess washbuffer and then air-dried for 5 minutes. 60 μL of elution buffer (5 mMTris-HCl pH 7.6), preheated to 70° C., was added to each well, the platewas incubated on a 90° C. hot plate for 5 minutes, and the eluate wasthen transferred to a fresh 96-well plate.

Cells grown on 100 mm or other standard plates may be treated similarly,using appropriate volumes of all solutions.

Total RNA Isolation

Total RNA was isolated using an RNEASY 96 (trademark) kit and bufferspurchased from Qiagen Inc. (Valencia, Calif.) following themanufacturer's recommended procedures. Briefly, for cells grown on96-well plates, growth medium was removed from the cells and each wellwas washed with 200 μL cold PBS. 150 μL Buffer RLT was added to eachwell and the plate vigorously agitated for 20 seconds. 150 μL of 70%ethanol was then added to each well and the contents mixed by pipettingthree times up and down. The samples were then transferred to the RNEASY96 (trademark) well plate attached to a QIAVAC (trademark) manifoldfitted with a waste collection tray and attached to a vacuum source.Vacuum was applied for 1 minute. 500 μL of Buffer RW1 was added to eachwell of the RNEASY 96 (trademark) plate and incubated for 15 minutes andthe vacuum was again applied for 1 minute. An additional 500 μL ofBuffer RW1 was added to each well of the RNEASY 96™ plate and the vacuumwas applied for 2 minutes. 1 mL of Buffer RPE was then added to eachwell of the RNEASY 96 (trademark) plate and the vacuum applied for aperiod of 90 seconds. The Buffer RPE wash was then repeated and thevacuum was applied for an additional 3 minutes. The plate was thenremoved from the QIAVAC (trademark) manifold and blotted dry on papertowels. The plate was then re-attached to the QIAVAC (trademark)manifold fitted with a collection tube rack containing 1.2 mL collectiontubes. RNA was then eluted by pipetting 140 μL of RNAse free water intoeach well, incubating 1 minute, and then applying the vacuum for 3minutes.

The repetitive pipetting and elution steps may be automated using aQIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia Calif.). Essentially,after lysing of the cells on the culture plate, the plate is transferredto the robot deck where the pipetting, DNase treatment and elution stepsare carried out.

Example 13 Real-Time Quantitative PCR Analysis of IGF-1R mRNA Levels

Quantitation of IGF-1R mRNA levels was accomplished by real-timequantitative PCR using the ABI PRISM (trademark) 7600, 7700, or 7900Sequence Detection System (PE-Applied Biosystems, Foster City, Calif.)according to manufacturer's instructions. This is a closed-tube,non-gel-based, fluorescence detection system which allowshigh-throughput quantitation of polymerase chain reaction (PCR) productsin real-time. As opposed to standard PCR in which amplification productsare quantitated after the PCR is completed, products in real-timequantitative PCR are quantitated as they accumulate. This isaccomplished by including in the PCR reaction an oligonucleotide probethat anneals specifically between the forward and reverse PCR primers,and contains two fluorescent dyes. A reporter dye (e.g., FAM or JOE,obtained from either PE-Applied Biosystems, Foster City, Calif., OperonTechnologies Inc., Alameda, Calif. or Integrated DNA Technologies Inc.,Coralville, Iowa) is attached to the 5′ end of the probe and a quencherdye (e.g., TAMRA, obtained from either PE-Applied Biosystems, FosterCity, Calif., Operon Technologies Inc., Alameda, Calif. or IntegratedDNA Technologies Inc., Coralville, Iowa) is attached to the 3′ end ofthe probe. When the probe and dyes are intact, reporter dye emission isquenched by the proximity of the 3′ quencher dye. During amplification,annealing of the probe to the target sequence creates a substrate thatcan be cleaved by the 5′-exonuclease activity of Taq polymerase. Duringthe extension phase of the PCR amplification cycle, cleavage of theprobe by Taq polymerase releases the reporter dye from the remainder ofthe probe (and hence from the quencher moiety) and a sequence-specificfluorescent signal is generated. With each cycle, additional reporterdye molecules are cleaved from their respective probes, and thefluorescence intensity is monitored at regular intervals by laser opticsbuilt into the ABI PRISM (trademark) Sequence Detection System. In eachassay, a series of parallel reactions containing serial dilutions ofmRNA from untreated control samples generates a standard curve that isused to quantitate the percent inhibition after antisenseoligonucleotide treatment of test samples.

Prior to quantitative PCR analysis, primer-probe sets specific to thetarget gene being measured are evaluated for their ability to be“multiplexed” with a GAPDH amplification reaction. In multiplexing, boththe target gene and the internal standard gene GAPDH are amplifiedconcurrently in a single sample. In this analysis, mRNA isolated fromuntreated cells is serially diluted. Each dilution is amplified in thepresence of primer-probe sets specific for GAPDH only, target gene only(“single-plexing”), or both (multiplexing). Following PCR amplification,standard curves of GAPDH and target mRNA signal as a function ofdilution are generated from both the single-plexed and multiplexedsamples. If both the slope and correlation coefficient of the GAPDH andtarget signals generated from the multiplexed samples fall within 10% oftheir corresponding values generated from the single-plexed samples, theprimer-probe set specific for that target is deemed multiplexable. Othermethods of PCR are also known in the art.

PCR reagents were obtained from Invitrogen Corporation, (Carlsbad,Calif.). RT-PCR reactions were carried out by adding 20 μL PCR cocktail(2.5×PCR buffer minus MgCl₂, 6.6 mM MgCl₂, 375 μM each of dATP, dCTP,dCTP and dGTP, 375 nM each of forward primer and reverse primer, 125 nMof probe, 4 Units RNAse inhibitor, 1.25 Units PLATINUM (registeredtrademark) Taq, 5 Units MuLV reverse transcriptase, and 2.5×ROX dye) to96-well plates containing 30 μL total RNA solution (20-200 ng). The RTreaction was carried out by incubation for 30 minutes at 48° C.Following a 10 minute incubation at 95° C. to activate the PLATINUM(registered trademark) Taq, 40 cycles of a two-step PCR protocol werecarried out: 95° C. for 15 seconds (denaturation) followed by 60° C. for1.5 minutes (annealing/extension).

Gene target quantities obtained by real time RT-PCR are normalized usingeither the expression level of GAPDH, a gene whose expression isconstant, or by quantifying total RNA using RiboGreen (trademark)(Molecular Probes, Inc. Eugene, Oreg.). GAPDH expression is quantifiedby real time RT-PCR, by being run simultaneously with the target,multiplexing, or separately. Total RNA is quantified using RiboGreen(trademark) RNA quantification reagent (Molecular Probes, Inc. Eugene,Oreg.). Methods of RNA quantification by RiboGreen (trademark) aretaught in Jones et al. (Analytical Biochemistry 265: 368-374, 1998).

In this assay, 170 μL of RiboGreen (trademark) working reagent(RiboGreen (trademark) reagent diluted 1:350 in 10 mM Tris-HCl, 1 mMEDTA, pH 7.5) is pipetted into a 96-well plate containing 30 μLpurified, cellular RNA. The plate is read in a CytoFluor 4000 (PEApplied Biosystems) with excitation at 485 nm and emission at 530 nm.

Probes and primers to human IGF-IR were designed to hybridize to theIGF-IR nucleotide sequence, using published sequence information(GenBank accession number NM000875 (FIGS. 2A and 2B), incorporatedherein as SEQ ID NO:76 or M69229 (SEQ ID NO:77) which is the 5′untranslated of the IGF-IR gene sequence). For human IGF-IR the PCRprimers were:

forward primer: CCCTTTCTTTGCAGTTTTCCC; (SEQ ID NO: 82 - ISIS 161212)reverse primer: CGTCGTCGGCCTCCATT; (SEQ ID NO: 83 - 161214) and the PCRprobe was: FAM- CCTTCCTGCCTCTCCGGGTTTGA-TAMRA (SEQ ID NO: 84 - ISIS161215)where FAM is the fluorescent dye and TAMRA is the quencher dye. Forhuman GAPDH the PCR primers were:

forward primer: GAAGGTGAAGGTCGGAGTC (SEQ ID NO: 94) reverse primer:GAAGATGGTGATGGGATTTC (SEQ ID NO: 95 and the PCR probe was:5′ JOE-CAAGCTTCCCGTTCTCAGCC- (SEQ ID NO: 95 TAMRA 3′where JOE is the fluorescent reporter dye and TAMRA is the quencher dye.

Example 14 Northern Blot Analysis of IGF-1R mRNA Levels

Eighteen hours after antisense treatment, cell monolayers were washedtwice with cold PBS and lysed in 1 mL RNAZOL (trademark)(TEL-TEST “B”Inc., Friendswood, Tex.). Total RNA was prepared followingmanufacturer's recommended protocols. Twenty micrograms of total RNA wasfractionated by electrophoresis through 1.2% w/v agarose gels containing1.1% v/v formaldehyde using a MOPS buffer system (AMRESCO, Inc. Solon,Ohio). RNA was transferred from the gel to HYBOND (trademark)-N+ nylonmembranes (Amersham Pharmacia Biotech, Piscataway, N.J.) by overnightcapillary transfer using a Northern/Southern Transfer buffer system(TEL-TEST “B” Inc., Friendswood, Tex.). RNA transfer was confirmed by UVvisualization. Membranes were fixed by UV cross-linking using aSTRATALINKER (trademark) UV Crosslinker 2400 (Stratagene, Inc, La Jolla,Calif.) and then probed using QUICKHYB (trademark) hybridizationsolution (Stratagene, La Jolla, Calif.) using manufacturer'srecommendations for stringent conditions.

To detect human IGF-IR an IGF-IR specific probe was prepared by PCRusing the forward primer for human IGF-IR CCCTTTCTTTGCAGTTTTCCC (SEQ IDNO:82-ISIS 161212) and the reverse primer for human IGF-IR reverseprimer sequence CGTCGTCGGCCTCCATT (SEQ ID NO:83-ISIS 161214). Tonormalize for variations in loading and transfer efficiency membraneswere stripped and probed for human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) RNA (Clontech, Palo Alto, Calif.). Hybridizedmembranes were visualized and quantitated using a PHOSPHORIMAGER(trademark) and IMAGEQUANT (tradeamrk) Software V3.3 (MolecularDynamics, Sunnyvale, Calif.). Data was normalized to GAPDH levels inuntreated controls.

Example 15 Antisense Inhibition of Human IGF-1R Expression

In accordance with the present invention, a series of antisensecompounds were designed to target different regions of the human IGF-IRmRNA or the 5′ untranslated region, using published sequences set forthin accession No. NM000875 (SEQ ID NO:76) and M69229 (SEQ ID NO:77). Thecompounds are shown in Table 1. “Target site” indicates the first(5′-most) nucleotide number on the particular target sequence to whichthe compound binds. All compounds in Table 1 are ASOs of either the 5′untranslated region or the coding region of the IGF-IR. The compoundswere analyzed for their effect on human IGF-IR mRNA levels byquantitative real-time PCR as described in other examples herein (seeFIG. 3 and Table 1). Data are averages from three experiments. Thepositive control for each datapoint is identified in the Table 1 bysequence ID number. If present, “N.D.” indicates “no data”.

As shown in Table 1, some lead compounds demonstrated at least someinhibition of IGF-IR expression in this assay and are thereforepreferred. Examples of preferred ASO's include ASO's ISIS 175308,175302, 175314, 175307, 175317, 175323, 323744, 323747, 323767, 323762and 323737. The target regions to which these preferred sequences arecomplementary are herein referred to as “preferred target segments” andare therefore preferred for targeting by compounds of the presentinvention. SEQ ID Nos 137 through 171 represent preferred targetsegments identified in IGF-1R. The “Target site” in Table 1 indicatesthe first (5′-most) nucleotide number on the particular target nucleicacid to which the oligonucleotide binds.

As these “preferred target segments” have been found by experimentationto be open to, and accessible for, hybridization with the antisensecompounds of the present invention, one of skill in the art willrecognize or be able to ascertain, using no more than routineexperimentation, further embodiments of the invention that encompassother compounds that specifically hybridize to these preferred targetsegments and consequently inhibit the expression of IGF-IR.

According to the present invention, antisense compounds includeantisense oligomeric compounds, ASOs, ribozymes, external guide sequence(EGS) oligonucleotides, alternate splicers, primers, probes, and othershort oligomeric compounds which hybridize to at least a portion of thetarget nucleic acid.

The purpose of this Example is to investigate the epidermal localizationof ASOs with full phosphorothioate 2′-O-(2-methoxy)ethyl gapmer (2′ MOEgapmer) or C5-propynyl-dU,dC-phosphorothioate (C5-propyne) chemistryfollowing topical application to psoriatic skin. Studies were performedon ex vivo psoriatic skin explants as shown in FIG. 1, with confocalmicroscopy, direct fluorescence and immunohistochemistry used to detectASO localization. In previous studies, an FITC conjugated C5-propyne ASOhas been shown to reach the basal layer of the epidermis after topicalapplication to psoriatic skin (White et al., Journal of InvestigativeDermatology 118: 1003-1007, 2002). In this Example, both 2′ MOE gapmerand C5-propyne ASOs were found to penetrate into the epidermis ofpsoriatic skin biopsies when formulated in either 5% w/v methylcelluloseor cream. ASOs of both chemistries seemed to accumulate in the basallayers of the epidermis as assessed by both direct fluorescencemicroscopy and immunohistochemical detection of ASOs. The localizationof FITC-ASOs was not obviously different from that of non-FITC ASOs.

Topical Application of ASOs

C5-propyne ASOs have been shown to accumulate in basal keratinocytes ofhuman psoriatic (but not normal) skin following topical application(White et al., 2002, supra), presumably due to the compromised barrierfunction of the stratum corneum in psoriasis. In addition, aphosphorothioate ASO was shown to accumulate in the basal keratinocytesof normal human skin when formulated in a cream (Mehta et al., J.Invest. Dermatol. 115: 805-812, 2000). A phosphorothioate-phosphodiesterhybrid ASO in distilled water failed to accumulate in basalkeratinocytes following topical application to human skin despiteappearing to cross the stratum corneum and accumulating in the cytoplasmof keratinocytes in the upper layers of the epidermis (Wingens et al.,Lab Invest. 79: 1415-1424, 1999).

The present Example investigated the localization of 2′ MOE gapmer ASOsin human psoriatic skin following topical application.

Oligonucleotides

The oligonucleotides employed are listed in Table 4.

TABLE 4 List of the four oligonucleotides used in topi- cal applicationstudies. Underlined sections bear 2′ MOE chemistry. De- tected Chem-Identifi- by 2E1 istry cation Sequence Ab C5 R451 UAACACGACGCGAAU-FITCUn- propyne [SEQ ID NO: 53] known 2′ MOE ASO FITC-TCCGTCATCGCTCCTCAGGGYes 251741 [SEQ ID NO: 54] 2′ MOE ASO TCCGTCATCGCTCCTCAGGG Yes 13920[SEQ ID NO: 55] 2′ MOE ASO FITC-TCCCGCCTGTGACATGCATT No 147979 [SEQ IDNO: 56]Collection of Psoriatic Skin Biopsies

Psoriatic skin biopsies were collected from volunteers. Up to three 8mm, full thickness, punch biopsies were collected from each volunteer bya dermatologist. The area from which biopsy is taken was not cleaned ordisinfected prior to biopsy collection. Biopsies were immediately placedon gauze (wetted with PBS) and stored on ice until used (˜2 hrs).

At the time of collection, the severity of psoriasis in the biopsies wasscored using the PRS (parameter rating scale) component of the PASI(psoriasis area severity index) score (Fredriksson et al., Dermatologica157: 238-244, 1978). In brief, erythema (redness), induration (swelling)and desquamation (flaking) were each scored from 0 (absent) to 4(severe) to give a PRS score of 0 to 12.

Live Confocal Microscopy

24 hrs after application of FITC-ASOs, biopsies were removed fromculture dishes and placed on coverslips, stratum corneum down, and adrop of PBS was placed on the exposed dermis to keep it moist. Liveconfocal microscopy was then performed as described previously (White etal., J. Invest. Dermatol. 112: 887-892, 1999; White et al., 2002,supra). In summary, topical application of FITC-ASO was assessed withexcitation at 488 nm (argon ion laser) and detection at 515 nm. Theinstrument used was an IX70 Olympus inverted microscope (OlympusAustralia, Melbourne, Australia) attached to an Optiscan f900e confocalsystem (Optiscan Pty, Melbourne, Australia).

Confocal microscopy results in sections en face to the surface of theskin and for each biopsy a series of images was taken at increasingdepth into the epidermis. Previous work indicates that fluorescence canbe detected up to 100 μm under the surface using this method.

In order to determine the epidermal location of FITC-ASO containingkeratinocytes, the criteria of White et al., (1999, supra) were used asa guide. These criteria were:

-   -   cellular morphology    -   presence and size of nuclei:        -   corneocytes anuclear        -   nuclei in keratinocytes of the stratum granulosum >15 μm        -   basal keratinocyte nuclei <10 μm    -   depth of cells (basal keratinocytes at least 50 μm below the        surface).        Processing of Tissue Samples

Following confocal microscopy, entire biopsies were fixed for 24 hrs in4% paraformaldehyde (4° C.) followed by 48 hrs in 0.5 M sucrose (4° C.).Biopsies were then submerged in graded ethanol (70%, 80%, 90% and 2×100%each for 90 min) followed by 2×90 min in limonene and 2×90 min inparaffin wax (65° C.) using a tissue processor (Shandon Citadel 1000,Shandon Inc, Pittsburgh, USA). Following processing, biopsies wereembedded in paraffin (Shandon Histocentre 2, Shandon Inc) and stored atroom temperature until required.

5 μm thick sections transverse to the epidermis were cut (Leica RM2035microtome, Leica Instruments, Wetzlar, Germany), transferred tosilane-coated glass slides and dried at 37° C. overnight. Sections werestored in a sealed container at room temperature until processed forhistological assessment of psoriasis, direct fluorescence orimmunohistochemistry.

Histological Assessment of Psoriasis

Sections (5 μm) from each psoriatic skin biopsy were de-waxed byimmersion in limonene for 2×5 min followed by consecutive 5 min washesin graded ethanol (100%, 90%, 80%, 70% and 50%) and 5 min in water.Sections were than stained with Harris' haematoxylin (stains cell nucleiblue) and eosin (stains cytoplasm and other tissue structures pink)before being washed in ethanol (2×15 sec) and limonene (2×15 sec).Sections were then cover-slipped with DPX mounting media (BDH LaboratorySupplies, Poole, England).

Detection of ASOs by Direct Fluorescence

Sections were de-waxed and washed as described above before beingcover-slipped with MOWIOL mounting media (Biosciences inc, La Jolla,USA) containing 2.5% DABCO anti-fade (Sigma, St Louis, USA). Imagebrightness was adjusted to correct for auto-fluorescence.Auto-fluorescence was defined as the fluorescence produced from vehicle(5% w/v methylcellulose or cream) treated sample.

Detection of ASOs by Immunohistochemistry (2E1 Ab)

Sections were de-waxed as described above and ASOs detected using theaffinity purified 2E1-B5 antibody (Berkeley Antibody Company, Berkeley,USA) supplied to us by Isis Pharmaceuticals. The 2E1-B5 antibody is amouse IgG1 that recognizes TGC and GC motifs in phosphorothioateoligonucleotides (Mehta et al., 2000, supra).

Sections were incubated in 1% v/v H₂O₂ in methanol for 30 min to quenchendogenous peroxidase activity, washed with PBS and incubated for 10 minin DAKO (registered trademark) ready-to-use proteinase K (DAKOcorporation, Carpenteria, USA). Sections were blocked with 1% w/v BSA/20ug/ml sheep IgG in PBS for 20 min before a 45 min incubation with the2E1 primary antibody (1/4000 dilution). Sections were again washed withPBS and the primary antibody detected using the Vectastain (registeredtrademark) Elite mouse ABC kit (Vector Laboratories, Burlingame, USA).The Vectastain (registered trademark) Elite mouse ABC kit uses asecondary biotinylated anti-mouse IgG that is then detected with anavidin and biotinylated horseradish peroxidase complex. DAB was used asthe substrate such that antibody localization was indicated by a browncoloration.

Image Capture

With the exception of confocal images (see ‘Live confocal microscopy’ inthis section), all images were captured using a Sony DXC-950P colourdigital camera (Sony, Tokyo, Japan) attached to a Nikon E600 microscope(Nikon Corporation, Tokyo, Japan) and controlled by a MCID M4 imagingsystem (Imaging Research Inc, St Catharines, Canada). Fluorescenceexcitation was provided by a Nikon HB-10103AF high-pressure mercury lamppower supply (Nikon Corporation) and viewed through an appropriatebarrier filter.

Assessment of Psoriatic Skin Biopsies

Psoriatic skin was collected from the abdomen, thigh, back, buttocks,shin, elbow or hips of volunteers. Up to three biopsies were taken fromeach individual and biopsies from each individual were allocated toseparate experimental groups.

The severity of psoriasis, as determined using the PRS, was 6.8±1.7(mean±SD, n=42) with a range from 3 to 9. The PRS was not significantlydifferent across experimental groups (p=0.9609, Kruskal-Wallisnon-parametric ANOVA).

Under histological examination, all biopsies appeared psoriatic althoughthere was considerable variation in morphology between biopsies. Inaddition to variations in the severity of psoriasis, the observedvariation may be due to the different body locations from which thebiopsies were taken. A thickened basal keratinocyte layer was visible inall biopsies, and in many (but not all) biopsies, elongated rete ridgesand cell nuclei in the stratum corneum (parakeratosis) were apparent.Cells resembling invading leukocytes were seen in the dermis of mostbiopsies.

Example 16 Topical Application of ASOs

To confirm the results of White et al., (2002, supra), whichdemonstrated localization of C5-propyne ASOs in basal keratinocytes ofpsoriatic skin biopsies, and to investigate the distribution of 2′ MOEASOs following topical application in 5% w/v methylcellulose or cream,the following FITC conjugated ASOs were applied to separate psoriaticskin biopsies:

-   -   0.1% w/w R451 (C5-propyne) in 5% w/v methylcellulose;    -   0.1% w/w ISIS 251741 (2′ MOE) in 5% w/v methylcellulose;    -   0.1% w/w ISIS 251741 (2′ MOE) in cream.

Direct fluorescence microscopy showed both the 2′ MOE gapmer ASO and theC5-propyne ASO in the epidermis of psoriatic skin lesions, withfluorescence clearly present in nuclei of basal keratinocytes.Fluorescence can also be seen in nuclei of cells that appear to beinvading leukocytes located in the dermis. There was no apparentdifference in the pattern of fluorescence produced by the 2′ MOE gapmerand C5-propyne ASOs following topical application.

Furthermore, 2′ MOE gapmer ASOs in cream showed an epidermaldistribution comparable to that see for 2′ MOE gapmer ASOs formulated in5% w/v methylcellulose, with no apparent difference in epidermallocalization of fluorescence.

These results were confirmed by live confocal microscopy which alsodemonstrated nuclear localization of FITC-AONs in cells fitting thecriteria for basal keratinocytes; cell nuclei <10 μm and least 50 μmbelow the surface). Interestingly, ASO appear to be in the nuclei ofparakeratotic corneocytes. The cells appear intermediate betweenkeratinocytes of the stratum granulosum and corneocytes, although theypresent at the surface of the epidermis. In some cases thesekeratinocytes appear to exclude ASO from their nuclei. Featuresconsistent with psoriasis were clearly observed which show eitherkeratinocytes of the stratum granulosum with nuclei much smaller thanwould be expected in normal skin, and/or basal keratinocytes much closerto the surface than would be expected in normal skin.

Example 17 Detection of a 5% ASO, Containing a 0.1% FITC-ASO Spike,Formulated in Cream

Higher ASO concentrations may be employed. Therefore, it is useful todetermine if an FITC-ASO contained as a 0.1% spike in a 5% total ASOformulation could be detected by direct fluorescence microscopy and/orconfocal microscopy. 2′ MOE FITC-ASO ISIS 251741 (0.1% w/w) mixed withthe non-FITC 2′ MOE ISIS 13920 (4.9% w/w) in cream was applied topsoriatic skin biopsies for this purpose.

Direct fluorescence and confocal microscopy images from samples treatedwith a 5% 2′ MOE containing a 0.1% FITC-ASO spike showed fluorescence inbasal keratinocytes. The increased concentration of ASO did not appearto alter the epidermal localization of fluorescence produced by theFITC-ASO, with localization similar to that observed following theapplication of 0.1% FITC-ASO alone.

Example 18 Benchmarking Antisense Oligonucleotides (ASOs)

Antisense oligonucleotides (ASOs) that target IGF-IR mRNA are proposedto be effective new therapeutic agents to reduce inflammatory and/orproliferative disorders. The purpose of this Example is to benchmarkthree preferred IGF-IR ASOs with full phosphorothioate “5-10-5,” 2′ MOEgapmer chemistry against DT1064 (SEQ ID NO:78), a 15 merC5-propynyl-dU,dC-phosphorothioate ASO. All C's and U's in DT1064 aresubjected to C5 propynylation. Studies were performed in a humankeratinocyte transfection system, with IGF-IR mRNA and protein levelsand cell proliferation as end-points. In previous studies, DT1064 hassuccessfully inhibited IGF-IR expression in this system (Wraight et al.,2000, supra; Fogarty et al., Antisense Nucleic Acid Drug Development 12:369-377, 2002).

The results show that the three IGF-IR ASOs reduced IGF-iR mRNA with thesame potency as DT1064. IGF-IR protein levels and cell proliferationrates were also reduced by the ASOs.

These findings support the use of the 2′ MOE gapmer chemistry forknockdown of IGF-IR mRNA. Based on its performance in the studiespresented in this Example, ASO 175317 is one 2′ MOE gapmer ASOparticularly useful for therapeutic trials.

2′ MOE Gapmers

The three “5-10-5,” 2′ MOE gapmers, phosphorothioate leads showedconcentration-dependent inhibition of IGF-IR mRNA in A549 cells (humanlung epithelial cells) as assessed by real-time PCR FIG. 3. The threeleads were assessed for activity in a human keratinocyte skin celltransfection system.

In Vitro Benchmarking of 2′ MOE Gapmers

The three lead ASOs have been “benchmarked” in vitro against DT1064 withthe following endpoints:—

-   1. Total IGF-IR mRNA assessed by real-time PCR;-   2. Total cellular IGF-IR protein determined by immunoblot;-   3. HaCaT keratinocyte cell growth rate assayed by amido black    dye-binding;    Oligonucleotides

Oligonucleotides used in this study are shown in Table 2.

TABLE 2 List of the seven oligonucleotides used for in vitro testing.Antisense/ Chemistry Identification Control 1C5-propynyl-dU,dC-phosphorothioate DT1064 A 2 (Abbreviation: C5-propyne)DT6416 C (mismatch) 3 R451 C (random) 4 2′-O-(2-methoxy)ethyl ISIS175314 A 5 5,10,5-gapmer, ISIS 175317 A 6 phosphorothioate throughoutISIS 175323 A 7 (Abbreviation: 2′ MOE gapmer) ISIS 129691 C (random) Thenucleotide sequences of the ASOs are present in FIG. 3.Cell Culture

Spontaneously immortalized human keratinocyte cell line, HaCaT (Boukampet al., 1988, supra) were used in this study. Cells were maintained asmonolayer cultures in Dulbecco's modified Eagle's medium (DMEM)supplemented with 10% w/v foetal calf serum (FCS) at 37° C. in anatmosphere of 5% V/V CO₂.

Transfection of Keratinocytes with Antisense Oligonucleotides

HaCaT keratinocytes (passage number 44 to 47) were seeded into the wellsof 96-well (real-time PCR), 24-well (cell proliferation) or 12-well(immunoblot or apoptosis) plates. 85-95% confluent cells were treatedwith the liposome preparation, Cytofectin GSV (GSV; Glen Research,Sterling Va., USA) alone, or complexed with antisense or controloligonucleotides. Untreated cells were also studied (untreated control).Each antisense or control oligonucleotide was diluted in serum-free DMEMto 20× the desired final concentration and mixed with an equal volume ofGSV (40 μg/ml). Lipid/oligonucleotide mixtures were allowed to complexat room temperature for 10 mins then diluted ten-fold with DMEMcontaining 10% w/v FCS. Cells were transfected with final concentrationsof 6.25, 25, 100 or 400 nM oligonucleotide and 2 μg/ml GSV.Transfections were performed in duplicate wells, while untreated andGSV-treated cells were run in four replicate wells.

IGF-IR mRNA levels

Total RNA was extracted using a RNEASY (registered trademark) Mini kit(Qiagen Inc., Valencia, Calif., USA) and 0.5 to 1 μg reverse transcribedusing the GeneAmp (registered trademark) RNA PCR kit (AppliedBiosystems, Foster City, Calif., USA), according to the manufacture'sinstructions. Semi-quantitative real-time PCR was used to determine theamount of IGF-I receptor mRNA in the sample relative to cells treatedwith GSV alone. Pre-developed reagents for the human IGF-I receptor(Applied Biosystems, Product No. 4319442F) and 18S (Product no.4319413E) containing primers and TaqMan (registered trademark)fluorescent probes were used in a multiplex PCR reaction tosimultaneously amplify both products in each sample. An ABI Prism(trademark) 7700 sequence detector (Applied Biosystems) was used for theanalysis. IGF-1R mRNA levels were then normalized to 18S. Twotransfection protocols were used—cells were transfected (1) once, 18 hbefore RNA extraction, or (2) a total of twice, at 24 and 48 h beforeRNA extraction.

IGF-1R Protein Levels

Following transfections with oligonucleotides every 24 h for three days,cell monolayers were washed with PBS, then lysed in a buffer containing50 mM HEPES pH 7.4, 150 mM NaCl, 1.5 mM MgCl₂, 10% v/v glycerol, 1% v/vTriton X-100, 100 ug/ml aprotinin. The total protein concentration ofthe lysates was assayed with the BCA Protein Assay kit (Pierce;Rockford, Ill., USA) which uses BSA as the protein standard. 25 or 30 μgof each lysate was resolved by SDS-PAGE (7% w/v acylamide) thentransblotted to Immobilon-P membrane (Millipore, Bedford,Massachusettes). Non-specific binding sites were blocked with 5% w/vskim milk powder then the filter probed with rabbit polyclonal IgGrecognizing the β-subunit of IGF-1R protein (C-20; Santa CruzBiotechnology Inc., Santa Cruz, Calif., USA). The IGF-1R-specific signalwas developed using the ECF western blotting kit (Amersham,Buckinghamshire, England, UK) and detected by chemifluorescence andphosphoimager scanning followed by quantification with ImageQuantsoftware (Molecular Dynamics, Sunnyvale, Calif., USA). Inter-filtervariation was controlled for by standardising signal intensities againstthe mean signal for cells treated with GSV alone.

Cell Proliferation Assay

Cells were grown to 40% confluence in 24-well plates and transfectedevery 24 h for up to 3 days. Cell number was determined at 0, 24, 48 and72 h using an amido black binding protocol in which binding of amidoblack to cellular protein (quantitated spectrophotometrically)correlates with cell number (Schultz et al., J. Immunol. Methods 167:1-13, 1994). Briefly, cell monolayers were fixed with 1% v/vglutaraldehyde in PBS then stained with 0.1% w/v amido black in Naacetate at pH 3.5 for 30 min. After a single wash in acidic H₂O, theprotein-bound dye was eluted with NaOH (50 mM) and the absorbance of theeluate monitored at 620 nm. Data are expressed relative to the signaldetermined for GSV-treated cells at 0 h.

IGF-1R mRNA

FIG. 4 shows the IGF-1R real-time PCR data for HaCaT keratinocytestreated with C5-propynes or 2′ MOE gapmers. The results were similarwhether cells were transfected once (FIG. 4A), or twice (FIG. 4B).IGF-1R mRNA levels were lower in cells transfected with DT1064, inkeeping with levels reported previously using RNase protection assays[Fogarty et al, 2002, supra]. All three lead ASOs also caused knockdownof IGF-1R mRNA. Furthermore, knockdown of the IGF-1R mRNA was similarfor the three ASO leads and DT1064. For example, in FIG. 4A, at 100 nMASO, the average reduction in mRNA was 68%, 77%, 75% and 78% for ASO175314, ASO 175317, ASO 175323 and DT1064, respectively.

IGF-1R Protein

FIG. 5A shows a representative IGF-I receptor western immunoblot ofHaCaT cells transfected with C5 propynes or 2′ MOE gapmers. The IGF-IRprotein (β chain) appears as a single band of molecular weight 110 kD.

The band intensities (expressed relative to cells treated with GSValone) from three separate experiments are combined and presented inFIG. 5B. The data show that DT1064 potently suppressed levels of IGF-IRprotein as shown previously (Fogarty et al., 2002, supra). Relative tocells treated with GSV alone, all three lead ASOs significantly reducedIGF-IR protein at 25 nM and 100 nM (P<0.01). ISIS 175317 and ISIS175323, but not ISIS 175314, knocked down IGF-IR protein at the 400 nMconcentration (P<0.01). There was no significant knockdown of IGF-IRprotein with ISIS 175317 or ISIS 175323 at the lowest concentration,6.25 nM.

Relative to the GSV control, transfection of HaCaT cells with DT1064provided apparent maximal reduction of 75% when cells were treated at aconcentration of 100 nM, while IGF-IR protein levels with ISIS 175317was approximately 60%. The knockdown of IGF-IR protein associated witheach of the ASOs is expressed as a percentage of its appropriatecontrol. This show that the ability of the ASOs to knockdown targetprotein is comparable to that of DT1064 (see Table 3).

TABLE 3 IGF-IR protein knockdown with ASOs expressed as a percentage ofcontrol olignonucleotides of the same chemistry at the sameconcentration. 6.25 nM 25 nM 100 nM 400 nM DT1064 (relative to 6416) 3448 59 41 DT1064 (relative to R451) 36 53 64 35 ISIS 175314 35 57** 51**30 ISIS 175317 28 60*** 65*** 50** ISIS 175323 19 50*** 58*** 44** *P <0.5, **P < 0.01, ***P < 0.001 versus IGF-1R protein in HaCaT cellstransfected with oligonucleotide of the same chemistry and dose; Tukey'stest.Cell Proliferation

The effect of IGF-1R-specific ASOs and control oligonucleotides on HaCaTproliferation is shown in FIG. 6. In untreated cells, keratinocyte cellnumbers increased more than four-fold over three days. GSV-treated cellsalso increased in number though not to the same extent as untreatedcells (64% of untreated at 72 h) suggesting some effect of the lipid onproliferation rates. Relative to untreated and GSV-treated cells, allcells treated with oligonucleotides showed lower rates of cellproliferation over the 3 days, with DT1064-treated cells having thelowest rates of cell proliferation at all time-points and at allconcentrations of oligonucleotide. Of the ASOs tested, there was a trendfor ISIS 175317 to be associated with the lowest rates of cellproliferation most notably at the 400 nM concentration.

Three IGF-OR lead ASOs have been tested in the HaCaT keratinocytetransfection system at MCRI. The major findings are:

All three ASO leads reduced IGF-IR mRNA levels compared with the GSVcontrol and the 2′ MOE gapmer random oligonucleotide. Relative toDT1064, the ASO leads gave a similar reduction in IGF-IR mRNA.

-   -   All three ASO leads significantly reduced IGF-IR protein        relative to the GSV control and the 2′ MOE random        oligonucleotide. The ASO leads reduced IGF-IR protein levels.        However, when expressed as a percentage of knock-down relative        to control oligonucleotides of the same chemistry, the effect of        the ASO leads was similar to that of DT1064.    -   All three ASO leads reduced cell proliferation rates relative to        the GSV control.        Ex Vivo Maintenance of Psoriatic Skin Biopsies

Biopsies were maintained for 24 hrs as described previously (Russo etal., Endocrinology 135: 1437-1446, 1994; White et al., 2002, supra).Briefly, subcutaneous fat was removed from the biopsies before they wereplaced, dermis down, on a BACTO (trademark) agar plug (Becton Dickinson,Franklin Lakes, USA) formed in the middle of a triangular stainlesssteel mesh. The steel mesh was designed to fit the centre well of a 60mm FALCON (registered trademark) centre-well organ culture dish (BectonDickinson) so that the agar plug was suspended over the centre well. Thecentre well was filled with Dulbecco's modified Eagle's medium(containing 10% w/v foetal calf serum, 50 IU/ml penicillin, 50 ug/mlstreptomycin) to the level of the agar plug and the outer well filledwith PBS to maintain humidity. Biopsies were incubated at 37° C. in anatmosphere of 5% v/v CO₂. FIG. 1 shows the tissue apparatus arrangement.

Example 19 Comparison of Direct Fluorescence Microscopy andImmunohistochemistry for Detection of ASOs

The 2′ MOE FITC-ASO ISIS 251741 was formulated at 0.1% w/w in 5% w/vmethylcellulose and applied topically to psoriatic skin biopsies. Bothdirect fluorescence and immunohistochemistry with the 2E1 antibody candetect ISIS 251741. This characteristic allowed the use of adjacentsections to directly compare ASO localization as determined by the twodetection technologies.

Both detection methods show a remarkably similar distribution of ASO.Both methods show accumulation of ASO in basal keratinocytes, exclusionof ASO from the nuclei of most keratinocytes of the stratum granulosum,and ASO in the nuclei of cells that appear to be invading leukocyteslocated in the dermis. Accumulation of ASO in the stratum corneum isapparent using both detection methods.

These results indicate that both direct fluorescence andimmunohistochemistry are viable methods for the detection of ASO inskin, however, both methodologies have their strengths and weaknesses.Digestion of skin sections with proteinase K (required beforeimmunohistochemistry) often resulted in degradation of tissuemorphology. Immunohistochemical detection of ASOs is also limited toASOs of specific chemistry (phosphorothioate) and nucleotide sequence(TGC or GC motifs) whereas any ASO can be conjugated to FITC.Furthermore, immunohistochemical detection of ASO appeared to be morevariable that detection of ASO by direct fluorescence. However,immunohistochemicaly stained sections can be stored and referred too fora longer period of time than fluorescent sections, which fade over time.In addition, there is lack of data concerning the effects of FITCconjugation on the physicochemical properties of ASOs.

Example 20 Effect of FITC Conjugation on Epidermal Localization of ASOsFollowing Topical Application

In order to determine whether an FITC tag attached to an ASO altersepidermal localization of the ASO following topical application,biopsies were treated with an ASO mixture containing the non-FITC-ASOISIS 13920 (detectable by immunohistochemistry but not directfluorescence) and the FITC-ASO ISIS 147979 (detectable by directfluorescence but not immunohistochemistry). Comparison of serialsections from tissues treated with this mixture showed no apparentdifference between the localization of the two ASOs. This data indicatesthat in psoriatic skin biopsies, an FITC tag on an ASO does not alterepidermal localization.

In order to control for the possibility that the FITC-ASO may effect theepidermal localization of ISIS 13920, biopsies were treated with 0.1%w/v ISIS 13920 alone. As can be seen, immunohistochemical detection ofISIS 13920 demonstrates a pattern of ASO distribution not apparentlydifferent to that seen for ASO 13920 mixed with ISIS 147979.

The localization of topically applied 2′ MOE ASOs in the epidermis ofpsoriatic skin lesions was investigated and compared with C5-propyneASOs. The major findings are:

-   -   Topically applied 2′ MOE gapmer ASOs in either 5% w/v        methylcellulose or cream were able to cross the stratum corneum        of psoriatic skin lesions. ASOs localized to the nuclei of basal        keratinocytes in the epidermis and invading leukocytes in the        dermis.    -   Epidermal localization following topical application does not        appear to differ between 2′ MOE gapmer and C5-propyne ASOs.    -   Following topical application, 2′ MOE gapmer ASOs can be        detected in the nuclei of basal keratinocytes by both direct        fluorescence microscopy (FITC conjugated ASOs only) and by        immunohistochemistry with the 2E1 Ab.    -   FITC conjugation of ASOs does not appear to alter their ability        to reach basal keratinocytes or their epidermal localisation        following topical application.

Example 21 Drug Formulation

Lyophilized ASOs were resuspended in sterile, distilled water and theconcentration of ASO determined by its optical density at 260 nm beforeformulation in either a 5% w/v methylcellulose gel or in a cream (IsisPharmaceuticals). The cream contained:

-   -   isopropyl myristate (10% w/w)    -   glyceryl monostearate (10% w/w)    -   polyoxyl 40 stearate (15% w/w)    -   hydroxypropyl methylcellulose (0.5% w/w)    -   monobasic sodium phosphate monohydrate (0.3% w/w)    -   dibasic sodium phosphate heptahydrate (0.9% w/w)    -   phenoxyethanol (2.5% w/w)    -   methylparaben (0.5% w/w)    -   propylparaben (0.5% w/w)    -   purified water (59.8% w/w)

For formulation in 5% w/v methylcellulose, 10% w/v methylcellulose (inPBS) was diluted two-fold with PBS containing ASOs at twice the desiredfinal concentration.

For formulation in cream, ASOs were dried (DNA Mini vacuum drier, Medoscompany, Melbourne, Australia) and then dissolved in an appropriateamount of cream to give the desired final concentration.

Example 22 Drug application

Approximately 30 min after biopsies were transferred to 37° C., ASOs orvehicle were weighed out (30 mg) and applied directly to anapproximately 4 mm diameter central region of biopsies with a smallspatula. A thin ring around the edge of the biopsy was kept free of ASOor vehicle in order to avoid application of ASO to the exposed edge ofthe sample. Despite these precautions, in approximately 20% of biopsiesASOs appeared to have been in contact with the edges of the biopsy asassessed by direct fluorescence microscopy.

Example 23 Experimental Groups

Pursuant of the aims of this Example, ASO formulations were applied toat least four biopsies from different individuals. The ASO formulationsand controls used were:

-   -   0.1% w/w R451 in 5% methylcellulose    -   0.1% w/w ISIS 251741 in 5% w/v methylcellulose    -   0.1% w/w ISIS 251741 in cream    -   0.1% w/w ISIS 251741 mixed with 4.9% w/2 ASO 13920 in cream    -   0.1% w/w ISIS 13920 in 5% w/v methylcellulose    -   0.1% w/w ISIS 147979 mixed with 0.1% w/w ASO 13920 in 5% v/v        methylcellulose    -   0.1% w/w ISIS 147979 in 5% w/v methylcellulose    -   5% w/v methylcellulose alone.        Cream alone.

Example 24 Benchmarking for ISIS 175317 and DT1064

This Example shows the benchmarking of human IGF-IR identified in aprimary screen against IGF-IR ASO (ISIS 175317) and DT1064 in HaCaTKeratinocytes.

IGF-IR mRNA levels were measured by real-time PCR after overnighttransfection of HaCaT keratinocytes with IGF-IR ASOs and controloligonucleotides. The results show that the four new leads, as well asISIS 175317 and DT1064, potently inhibited IGF-IR mRNA levels in aconcentration-dependent and sequence-specific manner. Relative to HaCaTcells treated with the transfection reagent alone, none of the fourrecently-identified IGF-IR ASOs suppressed IGF-IR mRNA levels with anygreater potency or efficacy than ISIS 175317.

Oligonucleotides

TABLE 5 List of the oligonucleotides used for in vitro testingAntisense/ Chemistry Identification Control 1 2′-O-(2-methoxy)ethyl ISIS175317 A 2 5,10,5-gapmer, ISIS 323737 A 3 phosphorothioate throughoutISIS 323744 A 4 All cytosine bases methylated ISIS 323762 A 5(Abbreviation: 2′ MOE gapmer) ISIS 323767 A 6 ISIS 306064 C (8nucleotide mismatch for ISIS 175317) 7 ISIS 129691 C (random) 8C5-propynyl-dU,dC-phosphorothioate DT1064 A 9 (Abbreviation: C5-propyne)DT6416 C (15 nucleotide mismatch for DT1064)

The concentration of each oligonucleotide was confirmed by its UVabsorbance at 260 nm prior to use.

Transfection of Keratinocytes with Antisense Oligonucleotides

HaCaT keratinocytes were maintained as monolayer cultures in Dulbecco'smodified Eagle's medium (DMEM) supplemented with 10% v/v foetal calfserum (FCS) at 37° C. in an atmosphere of 5% V/V CO₂/95% V/V O₂.

HaCaT keratinocytes [passage number 62 and 63 (FIG. 8) and 45 (FIG. 10)]were seeded into the wells of 96-well plates. 85-95% confluent cellswere treated with the liposome preparation, Cytofectin GSV alone, orcomplexed with antisense or control oligonucleotides for 20 h. Untreatedcells were also studied (untreated control). Each antisense or controloligonucleotide (20× final concentration) in serum-free DMEM was mixedwith an equal volume of GSV (20× final concentration; 40 μg/ml).Lipid/oligonucleotide mixtures were allowed to complex at roomtemperature for 10-15 min then diluted ten-fold with DMEM containing 10%v/v FCS. Cells were transfected with oligonucleotide (finalconcentration range, 0.4 to 200 nM) and 2 μg/ml GSV. Transfections wereperformed in duplicate wells, while untreated and GSV-treated cells wererun in four and six replicate wells, respectively.

IGF-I Receptor mRNA Levels

Total RNA was extracted using a RNeasy® Mini kit (Qiagen Inc., Valencia,Calif., USA) and approximately 0.1 to 0.2 μg were reverse transcribedusing the GeneAmp® RNA PCR kit (Applied Biosystems, Foster City, Calif.,USA), according to the manufacturer's instructions. Semi-quantitativereal-time PCR was used to determine the amount of IGF-I receptor mRNA inthe sample relative to cells treated with Cytofectin GSV alone.Pre-developed reagents for the human IGF-I receptor (Applied Biosystems,product no. 4319442F) and 18S ribosomal RNA (Product no. 4319413E)containing primers and TaqMan (Reg. Trademark) fluorescent probes wereused in a multiplex PCR reaction to simultaneously amplify both productsin each sample. An ABI Prism™ 7700 sequence detector (AppliedBiosystems) was used for the analysis. IGF-I receptor mRNA levels werethen normalised to 18S ribosomal RNA.

Biological Effects

The effect of antisense and control oligonucleotides on IGF-IR mRNAlevels were initially studied at concentrations of 6, 13, 25, 50, 100and 200 nM. These concentrations were chosen because they covered arange that would allow a comparison of data with previous in vitrobenchmarking experiments using 2′MOE gapmers in HaCaT keratinocytes.

FIG. 8 shows the IGF-I receptor mRNA levels for two experiments in whichHaCaT keratinocytes were transfected with 2′ MOE gapmers (ISIS 175317,ISIS 323737, ISIS 323744, ISIS 323762, ISIS 323767, ISIS 129691 or ISIS306064) or C5-proynes oligonucleotides (DT1064 or 6416). Relative toGSV-treated cells, all five 2′MOE gapmer ASOs potently suppressed IGF-IRmRNA levels (FIG. 9). Maximal target knockdown was similar for each2′MOE ASO (range 71-77%). Table 6 shows the maximum efficacy of theIGF-IR ASO leads.

In contrast, IGF-IR mRNA levels for the two 2′MOE gapmer controloligonucleotides (ISIS 129691 and ISIS 306064) were similar to thelevels of the GSV-treated cells, indicating that the effect of the 2′MOEgapmer ASOs on IGF-IR mRNA levels was sequence-specific (FIG. 8). Theeffect of DT1064 on IGF-IR suppression was maximal at 77% at 100 nM.However, transfection of HaCaT cells with the C-5 propyne mismatchcontrol oligonucleotide (6416) also suppressed IGF-IR mRNA levels by upto 46% at the same concentration. This is consistent with previous data.

Since maximal or near-maximal suppression of IGF-IR mRNA levels was seenwith the ASOs in the concentration range from 25 to 200 nM it wasdifficult to discriminate between the efficacy of the five 2′MOE ASOs.Therefore it was decided to test their effect at lower concentrations.FIG. 10 shows IGF-IR

TABLE 6 Maximum efficacy of ISIS IGF-IR ASO leads IGF-IR mRNA levels 2′MOE gapmer IGF-1R ASO (% cytofectin GSV-treated cells) ISIS 323762 23.1ISIS 323767 24.3 ISIS 175317 26.8 ISIS 323737 27.5 ISIS 323744 29.5Calculated from maximum efficacy IGF-IR mRNA levels (% of cytofectinGSV-treated cells) reported in FIG. 2.mRNA levels from a single experiment in which HaCaT cells were treatedwith oligonucleotides at 0.4, 1.6, 3, 6, 25, or 100 nM. As in theearlier experiments at the higher concentrations, all five 2′MOE ASOsspecifically suppressed IGF-IR mRNA levels, and did so with similarpotency. The maximal inhibition of IGF-IR mRNA was 87% for ISIS 175317,similar to that of the recently identified 2′MOE gapmer ASOs (range79-85% of GSV). The response to ASO treatment was concentrationdependent in this range. Treatment of HaCaT keratinocytes with 25 nMDT1064 suppressed IGF-IR mRNA levels by 81%, similar to the 2′MOE gapmerASOs.

FIG. 11 shows the concentration-response curves for the IGF-IR targeted2′MOE ASOs (same data as FIG. 10). With the possible exception of ISIS323767, these data indicate similar potencies of ISIS 175317 and the2′MOE gapmers. This is reflected in the EC₅₀ calculated from theconcentration-response curves and listed in Table 7. The EC₅₀ for DT1064was 3.2 nM (C.I. 2.1-4.7). It is important to note that the EC₅₀ valuespresented in Table 7 were calculated from a single experiment and aregiven as an estimate only. Additional concentration-response experimentswould be required to give a more accurate estimate of the EC₅₀ valuesfor the ASOs.

TABLE 7 Mean EC₅₀ (95% confidence intervals) for 2′MOE gapmer ASOsuppression of IGF-IR mRNA levels in HaCaT keratinocytes 2′MOE gapmerASO EC₅₀ [nM] ISIS 175317 2.6 (1.8-3.6) ISIS 323744 2.8 (2.2-3.6) ISIS323737 3.2 (0.4-25.8) ISIS 323762 3.3 (2.4-4.3) ISIS 323767 4.2(2.4-7.4)The concentration response experiments reported here were performedtwice at the higher concentration range and showed no difference inmaximum efficacy (Table 6). The concentration response experiment wasperformed once at the lower concentration range. Across all of theconcentrations studied (0.4 to 200 nM), none of the four leads used inthis Example appeared to exhibit greater IGF-IR mRNA knockdown than ISIS175317 in HaCaT keratinocytes (FIGS. 9 and 11). Examination of theconcentration-response curves showed similar potency between ISIS 175317and the four leads as assessed by EC₅₀ (FIG. 11).Antisense Oligonucleotide

Details of the IGF-IR ASO used in this Example are provided in Table 5.The underline nucleotides are 2′MOE modifications. All cytosine basesare methylated.

TABLE 5 IGF-IR ASO Chemistry Identification Sequence 2′MOE gapmer, ISIS175317 CGAAGGAAACAATACTCCGA phosphorothio- (SEQ ID ate throughout NO:125)ISIS 175317 (SEQ ID NO:125) was manufactured to research-grade qualityby Isis Pharmaceuticals, California, USA.Collection of Psoriatic Skin Biopsies

Psoriatic skin biopsies were collected from eleven volunteers underehtical conditions (Protocol 22023A of the Royal Children's HospitalEthics in Human Research Committee, Melbourne, Australia). Three 8 mm,full thickness, punch biopsies were collected from the same lesion ineach volunteer by a dermatologist. The area from which the biopsies weretaken was not cleaned or disinfected prior to biopsy collection.Biopsies were immediately plcated on guaze (wet with PBS) and stored onice until used (˜2h). At the time of collection, the severity ofpsoriasis in the biopsies was scored using the PRS (parameter ratingscale) component of the PASI (psoriasis area severity index) score(Fredriksson et al., 1978 supra). In brief, erthema (redness),induration (swelling) and desquanmation (flaking) were each scored from0 (absent) to 4 (severe) to give a PRS score of 0 to 12.

Ex Vivo Maintenance of Psoriatic Skin Biopsies

Biopsies were maintained for 24 h as previously described (Russo et al.,1994 supra; White et al., 2002 supra). Briefly, subcutaneous fat wasremoved from the biopsies and they were then placed, dermis down, on aBACTO™ agar plug (Becton Dickinson, Franlin Lakes, USA) formed in themiddle of a triangular stainless steel mesh. The steel mesh was designedto fit the center well of a 60 mm FALCON (Reg. Trademark) center-wellorgan culture dish (Becton Dickinson) so that the agar plug wassuspended over the center well. The center well was filled withDulbecco's modified Eagles medium (containing 10$ v/v foetal calf serum,501 U/ml penicillin, 50 ug/ml streptomycin) to the level of the agarplug and the outer well was filled with PBS to maintain humidity.Biopsies were incubated at 37° C. in an atmosphere of 5% v/v CO₂. Thetissue apparatus arrangement is shown in FIG. 1.

Drug Formulation

Lyophilised ISIS 175317 (SEQ ID NO:125) was resuspended in steriledistilled water and the concentration of the solution was determined byits optical density at 260 nm. For formulation in cream, ASO's weredried (DNA Mini vacuum drier, Medos Company, Melbourne, Australia) thendissolved in an appropriate amount of cream (Isis Pharmaceuticals, USA)to give a final concentration of 10% w/w. The cream contained:

-   -   isopropyl myristate (10% w/w)    -   pehnoxyethanol (2.5% w/w)    -   glyceryl monostearate (10% w/w)    -   methylparaben (0.5% w/w)    -   polyoxyl 40 stearate (15% w/w)    -   propylparaben (0.5% w/w)    -   hydroxypropyl methylcellulose (0.5% w/w)    -   purified water (59.8% w/w)    -   monobasic sodium phosphate monohydrate (0.3$ w/w)    -   dibasic sodium phosphate heptahydrate (0.9% 2/2)        Drug Application

After a 30-minute pre-incubation of the biopsy at 37° C., 30 mg ofpre-weighed drug or vehicle was applied directly to an approximately 4mm diameter central region of each biopsy with a small spatula. A thinring around the edge of the biopsy was kept free of ISIS 175317 (SEQ IDNO:125) or vehicle in order to avoid the cream touching the exposed edgeof the sample. Previous studies in this laboratory using FITCASOsapplied in this way, have shown that in approximately 20% of cases, ASOscontact the edges of the biopsy.

Experimental Groups

Three biopsies were collected from each volunteer. One of the biopsieswas treated with vehicle (Isis cream) and the other two with 10% ISIS175317 (SEQ ID NO: 125) in the cream. This treatment regimen allowspaired (vehicle-treated biopsy paired to the average of the two ISIS175317 (SEQ ID NO: 125)-treated biopsies) analysis of the data, andcontrol for possible confounding effects caused by inter-subjectvariations in IGF-IR mRNA levels. Two biopsies from each patient weretreated with ISIS 175317 (SEQ ID NO: 125) to increase the likelihood ofdetecting any drug effect.

Separation of Epidermis from Dermis

At the end of the treatment period (24 h), tissue samples were incubatedin 0.5 M EDTA (pH 7.4) at 60° C. for 90 sec to disrupt theepidermal-dermal junction and allow separation of the epidermis anddermis by blunt dissection (Dusserre et al., 1992 supra). The separatedepidermis and dermis were snap frozen in liquid nitrogen and stored at−70° C. until the RNA was extracted.

Measurement of IGF-IR mRNA Levels by Real-Time PCR

Tissues were mechanically crushed in a stainless steel mortar and pestlethat had been chilled in liquid nitrogen. Total RNA was extracted usinga Rneasy (Reg. Trademark) Mini kit (Qiagen Inc., Valencia, Calif., USA).Total RNA (100 to 700 ng) was reverse transcribed using the GeneAmp RNAPCR kit (Applied Biosystems, Foster City, Calif., USA), according to themanufacturer's instructions. The amount of starting RNA was matched asclosely as practicable for each set of biopsies and all samples werereverse-transcribed in the same reaction. Semi-quantitative real-timePCR was used to determine the amount of IGF-IR mRNA in biopsies relativeto 18S RNA. Pre-developed reagents for human IGF-IR (Applied Biosystems,product no. 4319442F) and 18S (product no. 4319413E) containing primersand TaqMan (Reg. Trademark) fluorescent probes were used in a multiplexPCR reaction to simultaneously amplify both products in each sample.Each sample was assayed in duplicate. An ABI Prism 7700 sequencedetector (Applied Biosystems) was used for the analysis. IGF-IR mRNAlevels were normalised to 18S.

Statistical Analysis

For statistical analysis, biopsies from each individual were paired.Each vehicletreated biopsy was paired to the average of the two ISIS175317 (SEQ ID NO:125)-treated biopsies. For comparison of IGF-IR mRNAlevels in ISIS 175317 (SEQ ID NO:125)-treated and vehicle-treatedbiopsies, a parametric paired t-test was used. This test assumes thatthe underlying population has a Gaussian distribution. These data didnot differ significantly (P>0.1) from that expected if sampling was froma population with a Gaussian distribution as assessed by a modifiedKolmogorov-Smirnov test (Dallal et al., 1986 supra). Furthermore, dotplots of the differences in IGF-IR mRNA levels between vehicle and ISIS175317 (SEQ ID NO: 125) treated biopsies appeared to be from apopulation with a Gaussian distribution. For all other comparisons, thenon-parametric Wilcoxon matched pairs test was used. This test makes noassumptions about the underlying population distribution. Statisticalanalysis was performed using GraphPad Prism version 3.00 for Windows(GraphPad Software, San Diego, Calif. USA). All data are presented asmean±one standard deviation.

Measurement of IGF-IR, GAPDH, HPRT, Insulin Receptor (IR), Caspase 3 &Bax mRNA Levels by Real-Time PCR

Tissues were mechanically crushed in a stainless steel mortar and pestlethat had been chilled in liquid nitrogen. Total RNA was extracted usinga Rneasy (Reg. Trademark) Mini kit (Qiagen Inc., Valencia, Calif., USA).Total RNA (100 to 700 ng) was reverse transcribed using the GeneAmp(Reg. Trademark). RNA PCR kit (Applied Biosystems, Foster City, Calif.,USA), according to the manufacturer's instructions. The amount ofstarting RNA was matched as closely as practicable for each set ofbiopsies and all samples were reverse-transcribed in the same reaction.

Semi-quantitative real-time PCR was used to determine the amount ofIGF-IR, GAPDH, HPRT, Insulin receptor (IR), Caspase 3 & Bax mRNA inbiopsies relative to 18S RNA. Pre-developed reagents for the abovementioned genes (Applied Biosystems, product # 4319442F (IGF-IR), #433764F (GAPDH), # 4333768F (HPRT), # 4318283F (Bax) and AppliedBiosystems ‘assay-on-demand’ assay ID # Hs00263337_ml (Caspase 3) and #Hs00169631_ml (Insulin receptor)) were each individually used in amultiplex PCR reaction with a pre-developed reagent for 18S (AppliedBiosystems, product no. 4319413E). These pre-developed reagentscontained primers and TaqMan (Reg. Trademark) fluorescent probes and,when used in a multiplex PCR reaction, simultaneously amplified thetarget gene (IGF-IR, GAPDH, HPRT, Insulin receptor (IR), Caspase 3 orBax) and 18S in each sample. Each sample was assayed in duplicate. AnABI Prism™ 7700 sequence detector (Applied Biosystems) was used for theanalysis. IGF-IR, GAPDH, HPRT, Insulin receptor (IR), Caspase 3 and BaxmRNA levels were normalised to 18S.

Statistical Analysis

For statistical analysis, biopsies from each individual were paired.Each vehicle-treated biopsy was paired to the average of the two ISIS175317 (SEQ ID NO: 125)-treated biopsies.

For comparison of the gene of interest's mRNA levels in ISIS 175317 (SEQID NO: 125)-treated and vehicle-treated biopsies, a parametric pairedt-test was used. This test assumes that the underlying population has aGaussian distribution. These data did not differ significantly (P>0.1)from that expected if sampling was from a population with a Gaussiandistribution as assessed by a modified Kolmogorov-Smirnov test (Dallalet al., 1986). Furthermore, dot plots of the differences in the gene ofinterest's mRNA levels between vehicle and ISIS 175317 (SEQ ID NO: 125)treated biopsies appeared to be from a population with a Gaussiandistribution.

Statistical analysis was performed using GraphPad Prism version 3.00 forWindows (GraphPad Software, San Diego, Calif. USA). All data ispresented as mean±one standard deviation).

The results are shown in FIGS. 12 to 14 and clearly demonstrate theeffiacy of the ISIS 175317 (SEQ ID NO:125) ASO in the cream to reduceIGF-IR and RNA in normal epiderms, dermis and psoriatic epidermis. Theresults in FIG. 14 clearly demonstrate the specificity of this ASO forIGF-IR.

Those skilled in the art will appreciate that the invention describedherein is susceptible to variations and modifications other than thosespecifically described. It is to be understood that the inventionincludes all such variations and modifications. The invention alsoincludes all of the steps, features, compositions and compounds referredto or indicated in this specification, individually or collectively, andany and all combinations of any two or more of said steps or features.

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1. A composition comprising an antisense compound consisting of amodified oligonucleotide consisting of 20 linked nucleosides andconsisting of a nucleobase sequence recited in SEQ ID NO: 125, whereinsaid compound inhibits the expression of IGF-IR, and wherein saidmodified oligonucleotide consists of a ten deoxynucleotide regionflanked on both the 5′ and 3′ end of said ten deoxynucleotide regionwith five 2′-O-(2-methoxyethoxy)nucleotides, wherein eachinternucleoside linkage in said modified oligonucleotide is aphosphorothioate linkage and each cytosine in said modifiedoligonucleotide is a 5-methylcytosine, said composition furthercomprising one or more pharmaceutically acceptable carriers and/ordiluents.
 2. A kit comprising the composition of claim 1 or an antisensecompound consisting of a modified oligonucleotide consisting of 20linked nucleosides and consisting of a nucleobase sequence recited inSEQ ID NO: 125, wherein said compound inhibits the expression of IGF-IR,and wherein said modified oligonucleotide consists of a tendeoxynucleotide region flanked on both the 5′ and 3′ end of said tendeoxynucleotide region with five 2′-O-(2-methoxyethoxy)nucleotides,wherein each internucleoside linkage in said modified oligonucleotide isa phosphorothioate linkage and each cytosine in said modifiedoligonucleotide is a 5-methylcytosine.